Identifying dormant growth state of mycobacteria by orthogonal

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Identifying dormant growth state of mycobacteria by orthogonal analytical approaches on a single cell and ensemble basis Anna-Cathrine Neumann, David Bauer, Michael Hölscher, Christoph Haisch, and Andreas Wieser Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03646 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018

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Analytical Chemistry

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Submission: Analytical chemistry

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Identifying dormant growth state of mycobacteria by orthogonal

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analytical approaches on a single cell and ensemble basis

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Neumann A-C1,2,3, Bauer D2,4, Hoelscher M1,3, Haisch C4*, Wieser A.1,2,3*

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1: Division of Infectious Diseases and Tropical Medicine, University Hospital, LMU Munich 2: Chair of Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Faculty of Medicine, LMU Munich 3: German Center for Infection Research (DZIF), Partner Site Munich, 80802 Munich, Germany 4: Chair of Analytical Chemistry and Water Chemistry, Technical University of Munich

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Prof. Dr. Christoph Haisch Chair of Analytical Chemistry, Technical University of Munich Marchioninistr. 17 81377 Munich, Germany [email protected] Tel +49 89 2180 78242 Fax +49 89 2180 99 78242

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Graphical Abstract

* Correspondence is to be directed to:

PD Dr. Andreas Wieser Division of Infectious Diseases and Tropical Medicine, University Hospital, LMU Munich Leopoldstr. 5 80802 Munich, Germany [email protected] +49 89 2180 78 296

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Tuberculosis is currently the single most deadly infectious disease in the world and a public health

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priority as defined by WHO. Although the disease is in general curable, treatment success is hampered

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by the necessity of a long and side effect prone treatment. Low treatment efficiency may be partly due

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to the special growth states mycobacteria enter to avoid being killed by antibiotics and to persist longer

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within the host. Such growth states have been recently defined as dormant or persistent. We produced

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dormant model-organism cultures using an acidification model and characterized those by a multi-

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layered approach using mass spectrometry (MALDI-TOF), microscopy (SEM, Raman), and

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microbiological techniques (CFU, OD600, ATP-levels). With a fast and 96-well-adapted extraction

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protocol, mycobacteria could be inactivated and extracted for MALDI-TOF analysis. For the first time,

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we demonstrate growth-state-dependent changes in the mass signatures of the culture, allowing for

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a reliable differentiation of dormant state and exponential growth. We also demonstrate resuscitation

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from dormant state back to exponential growth. Viable mycobacteria were immobilized and single

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organisms were analyzed individually by Raman microscopy. For single-cell Raman microscopy,

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Mycobacterium smegmatis cultures were fixed using a new fast and gentle single-step immobilization

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technique on a hydrophobic glass slide. We were able to distinguish single viable bacteria in the

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dormant state from their rapidly growing, genetically identical counterparts, identifying the growth

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state of the culture based on single-organism spectra. This allows for the separation of heterogeneous

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cultures depending on their growth state using the destruction-free optical method of Raman

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microscopy.

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Mycobacteria are the causative agent of the world’s most deadly infectious disease, tuberculosis.1

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Tuberculosis is an ancient disease with currently about 1.7 billion infected individuals all over the

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globe. With the development of antimycobacterial drugs, therapy has become available to many

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patients. The required treatment however, is of many months duration2 and associated with significant

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side effects.3,4 While over 90% of all mycobacteria are killed within the first two weeks of treatment, it

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still takes an additional 26 weeks to successfully cure the majority of patients. There is consensus

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amongst the research community that a prerequisite for identifying shorter treatments is a better

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understanding of the mechanisms of how subpopulations of mycobacteria evade treatment. In the last

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years, different growth states of mycobacteria were detected, among them, the so-called dormant

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state.5-7 This growth phase is understood now to be of prominent importance as it has been correlated

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with poor treatment outcome when detected in the sputum of patients under treatment.8,9 In vitro

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models could reproduce such dormant states10 and demonstrate less efficient killing of the bacteria by

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antimycobacterial drugs and slower metabolism than during rapid growth in full media.11 Mycobacteria

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also offer challenges regarding extraction of their constituents for analysis as well as immobilization

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for imaging, as they produce a highly hydrophobic and chemically resistant waxy cell wall. Besides,

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they exhibit remarkable tenacity, making inactivation protocols for analysis outside the BSL (bio-safety-

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level) 3 laboratory difficult.

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Mycobacterium smegmatis (M. smegmatis) can be used as a model organism to study its highly

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pathogenic relative, Mycobacterium tuberculosis (Mtb).12,13 This is especially interesting as it can even

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be used as a model for drug resistant tuberculosis14 and can also enter the dormant states of

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interest,15,16 while offering an overall faster growth and less biosafety concerns, being classified as BSL

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2 in Germany and BSL 1 in many other parts of the world.

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Experimental section

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Bacterial strains and cultivation 4 ACS Paragon Plus Environment

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To generate dormant cultures, M. smegmatis (strain MC2 155 17) was initially cultivated at 37°C, under

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agitation at 140 rpm for 24h in nutrient broth medium, containing 8.0 g nutrient broth per liter. The

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following day, 1 mL of this culture was added to 100 mL of Sautonˈs medium, containing per liter:

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K2HPO4, 0.18 g; MgSO4·7H2O, 0.166 g; NaH2PO4, 0.056 g; L-asparagine, 1.33 g; glycerol, 20 ml;

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ferricammonium citrate, 0.017 g; citric acid, 0.66 g; polysorbate 80, 0.833 g and water. The pH was

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adjusted to 6.0 using 1 M HCl and the final media was supplemented with 0.05% Tween-80.15 The

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culture was subsequently incubated for 20 days at 37°C under agitation at 140 rpm. Reference wild-

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type cultures were cultivated overnight (37°C, 140 rpm) in M7H9 media (supplemented with OADC

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and 0.05% Tween-80). Resuscitation of dormant M. smegmatis cultures was performed with two

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different protocols. First, 100µl dormant culture was directly inoculated into 25ml of supplemented

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M7H9 media (OADC, 0.05% Tween-80). Second, 1ml of dormant culture was centrifuged (3500rpm,

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20min at 20°C), the pellet washed once in 1ml M7H9 media to remove residual old media and

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resuspended again in 1ml of new M7H9 media. After a second centrifugation step, 250µl of the washed

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culture was used to inoculate 25ml of supplemented M7H9 media (OADC, 0.05% Tween-80). All

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resuscitated cultures were incubated for at least 24h at 37°C under agitation at 140rpm.

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ATP Determination

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Bacterial cultures were harvested at distinct time points and chilled on ice until centrifugation (10000

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rpm 10 min 4°C) to remove excess media. The pellet was resuspended in 500µl of NaCl 0.9% normal

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saline and lysed using a bead beater FastPrep-24 (MP Biomedicals, USA) with 200µl 0.1mm diameter

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zirconia beads (Sigma-Aldrich, Munich, Germany) for 45s at 7.5m/s speed setting with intermediate

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cooling on ice. Homogenates were divided and 5µl of each sample was used for ATP measurements

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with the ATP determination Kit (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s

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instructions using the TriStar LB941 (Berthold Technologies, Bad Wildbad, Germany) reader machine.

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Samples were kept on ice for all the time, to avoid premature decay of ATP within the samples. 5 ACS Paragon Plus Environment


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Bacterial cells were enumerated with the CyFlow Cube 6 flow cytometer (Sysmex Deutschland,

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Norderstedt, Germany) for calculation of average cellular ATP content.

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MALDI-TOF MS sample preparation

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The extraction of mycobacterial proteins was performed using filter plates (0.22 µm pore size,

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hydrophilic polypropylene - GHP) placed on a vacuum manifold. The bacterial culture (300 µL) was

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added to the well without vacuum supply. Bacterial cells were then immobilized onto the filter

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membrane by applying vacuum until the growth medium was completely sucked through the

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membrane. After three consecutive washing steps with 300 µL dest. H2O with incubation time of 5 min

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each, the filter plate was placed on ice and 100 µL of ice-cold acetone was added for 5 min. Vacuum is

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used to remove the acetone through the chemically stable filter. To evaporate residual acetone sticking

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to the bottom of the plate, the filter plate was placed in a 37°C incubator for 7 min on a filter paper.

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Elution of the extracted protein was conducted by adding 20 µL of elution solution (50% acetonitrile,

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35% formic acid, 15% dest. H2O, v/v) to each well. After 5 min incubation at room temperature, a 96-

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well collection plate was placed below the filter plate and centrifuged at 4000 rpm for 3 min at 20°C

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to collect the extracts.

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MALDI-TOF MS analysis

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1µl of extract was directly spotted onto a polished steel MSP-96 MALDI target (Bruker Daltonik GmbH,

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Bremen, Germany). Dried spots were overlaid with 1 µL α-cyano-4-hydroxy-cinnamic acid (HCCA,

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10 mg/mL in 50% acetonitrile, 47.5% dest. H2O and 2.5% trifluoroacetic acid, v/v). After complete

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crystallization of the matrix, the measurements were performed on a Microflex LT benchtop mass

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spectrometer equipped with a 60 Hz nitrogen laser and controlled by FlexControl software (version:

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3.4.135.0, Bruker Daltonik GmbH, Bremen, Germany). Spectra were recorded in the positive linear

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mode. The parameter settings were optimized for the mass range between 2000 and 20000 Da and

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were as followed: ion source 1: 0 kV, ion source 2: 18.25 kV, pulsed ion extraction time: 130 ns, gain

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factor: 9.3x. For instrument calibration an external standard was chosen consisting of masses in the 6 ACS Paragon Plus Environment

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range between 3637.8 and 16952.3 Da (bacterial standard, BTS, Bruker Daltonik GmbH, Bremen,

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Germany). All acquisitions were recorded automatically by the instrument software; the final data are

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based on averaging 240 satisfactory shots in 40 shot steps.

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SEM sample preparation

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Formalin fixation was achieved by adding formalin to the bacterial culture with a final concentration

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of 3.7% followed by centrifugation for 5 min at 4500 rpm and 20°C. The cell pellet was resuspended in

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3.7% formalin solution. 10 µL fixed bacterial solution were spotted on stretched 0.7 mm aluminum foil

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and dried slightly at 37°C, just until the visible water film evaporated. Dehydration of the cells was

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subsequently performed using increasing concentrations of ethanol (10%, 30%, 70%, and 100% v/v,

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7 min each) the sample was finally dried at room temperature and stored in a desiccator until electron

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microscopy measurement.

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SEM analysis

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Samples were imaged on a scanning electron microscope (Zeiss Sigma VP Field Emission Scanning

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Microscope, Carl Zeiss Microscopy GmbH, Germany) under high vacuum, with a 3.1 mm working

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distance and 30 µm objective lens aperture. Images were collected using the secondary electron

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detector, voltage of 3kV. Length of bacteria was analyzed by manual evaluation, using Image J18 on the

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electron microscopic images. The pixel aspect ratio was determined using the scale bar tool followed

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by marking the clearly distinguishable bacteria with the ‘draw line’ and ‘measure size’ function.

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Raman sample preparation

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500 µL of a M. smegmatis culture (wild-type or dormant) were centrifuged for 4 min at 5000 g and 4°C,

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the supernatant discarded, and the cell pellet was resuspended in 500 µL 0.5% NaCl solution. After

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brief vortex mixing, the sample was centrifuged for a second time and the pellet finally resuspended

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in 250 µL 0.9% NaCl solution. For the fixation of live Mycobacteria on glass slides, no standardized

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techniques exist. In Raman microscopy and microscopic analysis in general, usually chemical fixation is 7 ACS Paragon Plus Environment


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employed or bacteria are fixed by drying.19 In both cases the cells are killed sooner or later. To allow

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observation of viable microorganisms a typical fixation is realized by optical tweezing or with surface

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modified slides.19,20 However, these methods request either a complex instrumentation or because of

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the complex and hydrophobic cell wall of Mycobacteria, no efficient binding can be obtained. For

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biosynthetic reactions using live Mycobacteria, hydrophobic silicone rubbers have been used to

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immobilize Mycobacteria.21 Similarly, we also exploited the hydrophobic nature of the mycobacteria

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for microscopic imaging by using commercially available hydrophobic glass slides (Paul Marienfeld

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GmbH, Lauda-Konigshofen, Germany). The bacteria are easily fixed by dipping the glass slide into the

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bacterial suspension. This sample preparation not only allows for a fast and reliable fixation of

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mycobacteria under physiological conditions, but also allows for a Raman analysis via an oil immersion

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objective, allowing for a high numerical aperture (NA = 1.4, working distance 0.15 mm). After

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positioning the glass slide with the fixed bacteria onto the microfluidic measurement chamber, it was

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immediately filled with physiologic NaCl solution (0.9%) and sealed with wax to avoid drying out of the

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chamber during the measurement.

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Raman measurements and data acquisition

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The Raman spectra were recorded using a WITec ALPHA300 R system (WITec GmbH, Ulm, Germany),

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equipped with a Cobolt DPL 532 nm solid state laser (Cobolt AB, Solna, Sweden), a truepower module

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ensuring a stable laser power throughout the whole experiments, and employing a 63x oil immersion

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objective (Zeiss C Plan-Apochromat 63x1.4 Oil; Carl Zeiss AG, Germany). A spectrometer grating with

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600 l/mm was used and detection was carried out with a Newton 970 EMCCD camera (Andor

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Technology Ltd., Belfast, UK). The spectra were acquired from single bacterial cells applying 10 mW

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laser power, 5 s integration time, and three accumulations, recording a spectral range from 200 to

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3200 cm-1. Spectra with a sufficient signal-to-noise ratio were selected and processed with MATLAB


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2018 (The MathWorks Inc, USA), as were all data handling steps presented here. Background

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subtraction and normalization were carried out by the functions msbackadj and msnormalize.

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Results and Discussion

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For the in vitro generation of dormant mycobacteria, different models can be employed. The hypoxic

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model published by Wayne et al. uses a long-term culture with gradual oxygen depletion and starvation

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through nutrient depletion.22,23 In contrast, the acidification model is based on the fact that

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mycobacteria are sensitive to low pH values and can survive in the macrophage phagosomes due to

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arresting acidification.15,24.The big advantage of this protocol is the possibility to use it for a wide range

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of mycobacterial strains and the samples can be handled under regular atmospheric conditions. Thus,

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this protocol was used throughout the study.

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M. smegmatis was initially grown for 24 h in M7H9 medium supplemented with OADC and Tween-80.

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The inoculum (1 mL) was added to 100 mL of Sauton's medium. The initial pH value was 6.0 and during

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an growth period of 21 days the medium underwent further gradual acidification (pH of 4.0-4.5), as a

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result of bacterial metabolism and release of metabolites (e.g. organic acids).15 To verify the dormant

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state, commonly used techniques in the field were applied.25,26

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To assess the ability of the dormant culture to regrow, dormant M. smegmatis cultures were

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resuscitated by inoculation of a small amount of dormant bacteria into supplemented M7H9 media.

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Two different protocols were chosen as detailed above. In brief, washed and unwashed dormant

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bacterial cells were regrown for at least 24h and showed robust growth under both conditions.

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Examination of dormant state

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Wild type,dormant as well as regrown/resuscitated cultures were examined microscopically. The size

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and morphological appearance of the cells differed between wild-type/resuscitated and dormant

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bacteria. Smaller, odd-shaped growth forms and clumping cells could be observed as has been 9 ACS Paragon Plus Environment


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described for dormant mycobacteria in literature.27 To characterize in more detail the changes in

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bacterial growth form and shape, electron microscopy was performed. The electron microscopic

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images (Figure 1 A and B) confirm the changes in shape observed by optical microscopy and

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demonstrate a pearl-sting-like appearance of the dormant bacteria as opposed to the smooth and even

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shape of the fast growing wild-type organisms in rich media. Bacterial cells resuscitated from dormancy

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for >24h re-gain the elongated shape of exponentially growing cultures (Figure 1 A and C).

Figure 1: Scanning electron microscopy (SEM) images of M. smegmatis wild-type growth (A), M. smegmatis dormant state (B) and M. smegmatis cells resuscitated from dormant state (C).


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Figure 2: Bacterial length distribution determined by SEM, M. smegmatis wild-type (dark green, n=90), M. smegmatis dormant (red, n=117). Significant difference in length between the groups was observed (Whitney-Mann p

Identifying dormant growth state of mycobacteria by orthogonal

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