AAC Accepted Manuscript Posted Online 16 February 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.01380-15 Copyright © 2016 Turapov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
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Phenotypically adapted Mycobacterium tuberculosis populations from
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sputum are tolerant to first line drugs
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Obolbek Turapov1, Benjamin D. O’Connor1, Asel A. Sarybaeva1, Caroline
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Williams1, Hemu Patel2, Abdullaat S. Kadyrov3, Akpay S. Sarybaev4, Gerrit
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Woltmann5, Michael R. Barer1, 2, Galina V. Mukamolova1*.
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1
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Leicester, LE1 9HN, UK; 2Empath Pathology Services, Department of Clinical
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Microbiology, University Hospitals of Leicester NHS Trust, Leicester, UK
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3
Department of Infection, Immunity and Inflammation, University of Leicester,
National Center of Phthisiology, Akhunbaeva 90A, Bishkek, Kyrgyzstan; 4
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Kyrgyz Indian Mountain Biomedical Research Center, Togolok Moldo 3,
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Bishkek, Kyrgyzstan; 5Department of Respiratory Medicine, Glenfield
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Hospital, Leicester, UK
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*To whom correspondence should be addressed. E-mail:
[email protected]
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Running title: Phenotypically tolerant Mtb from sputum
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1
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ABSTRACT
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Tuberculous sputum contains multiple Mycobacterium tuberculosis (Mtb)
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populations with different requirements for isolation in vitro. These include
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cells forming colonies on solid media (platable Mtb), cells requiring standard
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liquid
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supplementation of liquid medium with culture supernatant for growth (SN-
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dependent Mtb). Here we describe protocols for the cryopreservation and
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direct assessment of antimicrobial tolerance of these Mtb populations within
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sputum. Our results show that first line drugs achieved only modest cidal
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effects on all three populations over 7 days (1-2.5xlog10 reductions) and SN-
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dependent Mtb were more tolerant to streptomycin and isoniazid compared to
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platable and non-platable Mtb. Susceptibility of platable Mtb to bactericidal
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drugs was significantly increased after passage in vitro, thus tolerance
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observed in the sputum populations was likely associated with mycobacterial
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adaptation to the host environment at some time prior to expectoration. Our
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findings support the use of a simple ex vivo system for testing drug efficacies
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against mycobacteria phenotypically adapted during tuberculosis infection.
medium
for
growth
(non-platable
Mtb),
and
cells
requiring
33
2
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INTRODUCTION
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Tuberculosis (TB) remains a major infectious disease that claims nearly two
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million lives annually (1). Both the requirement for prolonged treatment with a
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combination of drugs and the rise of multiple and extensively drug-resistant
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strains emphasize the need for effective new drugs. Mycobacterium
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tuberculosis (Mtb) adopts multiple physiological states during infection and
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this provides part of the rationale for use of multiple drugs (2).
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Sputum samples from patients are widely used for the microbiological
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diagnosis of TB by microscopy, culture and molecular techniques (3); they
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also represent a valuable source of mycobacteria phenotypically adapted to
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the in vivo environment. Sputum mycobacteria display a “fat and lazy
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persister-like” phenotype with reduced metabolic activity, prominent lipid
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bodies and a unique transcriptomic signature suggesting adaptation to slow-
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or non-growth (4). Application of growth assays allowed us to establish that
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mycobacterial populations in sputum produce distinct patterns of growth
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referred to here as “platable” (colony forming unit-producing), “non-platable”
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(liquid culture-dependent) (5) and “Rpf-dependent” (growth dependent on
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liquid medium supplemented with recombinant resuscitation-promoting factor
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(Rpf) or Rpf-containing culture supernatant (SN) (6).
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These distinct growth phenotypes may reflect the different in vivo
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populations that must be eliminated by chemotherapy and may also underpin
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well-known phenomena such as early bactericidal activity and relapse after
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treatment. Specific aspects of the in vivo environment are likely to be the
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major factors driving phenotypic heterogeneity (7, 8). Therefore it may be
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critical to assess activities against in vivo adapted bacilli to understand and
3
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predict the different responses of Mtb subpopulations to drug treatment.
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Mouse infection models have been used extensively to assess anti-TB drugs,
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however, they do not precisely represent the human in vivo environment and
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have certain well-recognized limitations (9).
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We propose that direct study of Mtb cells in sputum provides an important
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opportunity to demonstrate aspects of the chemotherapeutic efficacy of drugs
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and thereby facilitate drug development. The main challenge in direct
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assessment of sputum Mtb response to drugs is posed by the difficulty in
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reliable preservation of heterogeneous Mtb populations for multiple assays.
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While freezing and storage of in vitro grown Mtb cultures at -70-80°C did not
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influence mycobacterial viability in several studies (10-12), relatively little is
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known about viability of Mtb in sputum subjected to freezing. Tessema et al
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showed that storage of smear positive sputum samples at -20°C for up to 180
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days sustained an overall culture positivity rate of 90% on multiple media (13),
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but these authors did not compare bacterial numbers before and after
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storage. Other sputum preservation methods such as addition of 1%
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cetylpyridinium chloride also have certain limitations and influence Acid-Fast
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staining properties (14).
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In this proof-of-concept study we have developed a simple procedure for
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cryopreservation of heterogeneous Mtb populations from sputum and applied
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this to establish a novel direct drug tolerance assay using ex vivo
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mycobacteria from sputum.
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METHODS
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Patients
4
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The study was approved by Leicestershire, Northamptonshire and Rutland
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Research Ethics Committee (07/Q2501/58). Consenting patients who
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produced smear-positive sputum provided samples for the study before onset
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of chemotherapy. The presence of Mtb in all samples was confirmed by
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microscopy and culture. The identity of Mtb strains and their drug
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susceptibility profiles were determined as described below. Altogether
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samples from 15 patients have been used in this study. Samples were
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collected in Leicester (UK) and Bishkek (Kyrgyzstan). Sputa were kept at 4°C
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for up to 7 days before decontamination and freezing as previously described
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(6).
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Bacterial strains
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Mtb H37Rv was grown in 7H9 medium supplemented with 10% (v/v) OADC
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(Becton, Dickinson and Company), 0.05% (w/v) Tween 80 and 0.2 % (v/v)
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glycerol for production of culture supernatant and antimicrobial tolerance
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assays. This medium is referred to as supplemented 7H9 medium. Mtb
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isolates from sputum were grown on 7H10 agar or in 7H9 supplemented
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medium. Bacteria were grown in Sterilin™ Polypropylene 30mL Universal
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Containers with 5 ml of medium or Falcon™ 50mL Conical Centrifuge Tubes
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with 10 ml of medium or in 2 l Nunc™ Roller Bottles with 200 ml of medium.
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Tubes were incubated at 37°C with shaking (100 rpm). Mycobacterial strains
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were typed using MIRU-VNTR (15) as described previously (6).
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Preparation of culture supernatant
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Mtb H37Rv was cultured in supplemented 7H9 medium to an optical density of
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0.6-0.8 measured at 580 nm. Cultures were centrifuged at 3000xg for 20 min
5
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and supernatant was double filtered using 0.20 μm VWR® Filtration Systems.
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Aliquots of 10 or 25 ml were freeze-dried using Freeze drier AdVantage Plus
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ES-53 (Biopharma Process Systems Ltd, UK). Freeze-dried supernatant
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preparations were kept at -80°C for up to 6 months. As shown in Figure S1
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freeze-drying preserved resuscitation activity. For experiments freeze-dried
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supernatant was reconstituted in sterile deionized water and kept on ice for 30
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min to allow complete dissolving of the dried material. The dissolved
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supernatant was kept on ice and used within 1 hour.
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Decontamination and storage of sputum samples
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Sputum samples were decontaminated as described previously (6). Briefly,
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one volume of sputum was mixed with one volume of of 4% w/v NaOH and
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incubated at room temperature for 15 minutes. The mixture was neutralised
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with 2 volumes of 14% w/v KH2PO4. Decontaminated samples were
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centrifuged at 4000xg for 20 min, resuspended in the original volume of 10 %
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(v/v) sterile glycerol, phosphate buffered saline (PBS), or 200 mM trehalose.
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The mixture was passed through a 23 G blunt needle (Harvard Apparatus,
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UK) 5 times to maximize homogeneity and disperse potential mycobacterial
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aggregates (16, 17). The homogenized decontaminated Mtb from sputum
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samples were frozen and stored at -80°C in 0.5 ml aliquots. Sputum is often a
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viscous and clumpy material and this may create an unequal distribution of
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mycobacteria leading to a high variability between counts. To address this we
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introduced the additional step for the homogenization of sputum by passing
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the treated sample through blunt fine gauge needles. Application of this
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method ensures minimal variation of bacterial counts between frozen aliquots
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of sputum samples (Fig. S2).
6
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Growth assays
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Previous studies revealed three Mtb populations with different conditions for
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cultivation: (I) “Platable” populations grow on solid agar and numbers of
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platable Mtb are determined by counting colony-forming units (CFU). (II) “Non-
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platable” populations grow in liquid medium and their numbers assessed by
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Most-Probable Number assays in liquid 7H9 medium (MPN). (III) “Supernatant
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(SN)-dependent” Mtb can only grow in liquid medium supplemented with Rpf-
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containing supernatant and they are enumerated by MPN assay in 7H9 liquid
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medium supplemented with Rpf-containing culture supernatant (MPN_SN) (6).
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For CFU counts mycobacteria were serially diluted in supplemented 7H9
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medium and plated on 7H10 agar (Becton, Dickinson and Company). MPN
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counts were assessed in supplemented 7H9 medium and MPN_SN were
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done in 7H9 supplemented with 50% (v/v) culture supernatant. The BBL™
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MGIT™ PANTA™ antibiotic mixture (Becton, Dickinson and Company) was
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added to all media used for growth assays as recommended by the
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manufacturer. Agar and MPN plates were incubated at 37°C for up to 12
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weeks. MPN counts were calculated as described previously (6).
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Drug tolerance assays
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Tolerance for each drug was assessed using four sputum samples from
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separate patients. These samples are designated as S6, S19, S20 and S21.
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Each sample was decontaminated, homogenized and frozen in aliquots as
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described above. Decontaminated stored sputum samples (0.5 ml aliquots)
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were defrosted at room temperature and centrifuged for 20 min at 4000xg.
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The supernatant was discarded to minimize possible effects of the storage
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medium on Mtb responses to drug treatment and each pellet was
7
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resuspended in 0.5 ml of supplemented 7H9 medium. Falcon tubes containing
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10 ml of supplemented 7H9 medium with PANTA and an appropriate drug
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were inoculated with 0.5 ml of resuspended pellet. Antimicrobials were added
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to achieve the following final concentrations (μg/ml): streptomycin 10 and 20,
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rifampin 1 and 5, ethambutol 10 and 20, isoniazid 1 and 10, pyrazinamide 40
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and 100. Pyrazinamide was tested at pH 5.6 and 6.8; appropriate controls
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were done in both cases. Tubes were sealed and incubated at 37°C with
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shaking (100 rpm). Mtb CFU, MPN and MPN_SN counts were determined at 0
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(sample without drugs only), 3 and 7 days of incubation. At 3 days half of the
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incubated sample (5 ml) was transferred into a separate Falcon tube and
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centrifuged at 4000xg for 20 minutes. Supernatants were discarded and
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pellets were resuspended in 0.5 ml of 7H9 medium; the resuspended pellets
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were used for determination of viable counts. Experiments were done in
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duplicate. Drug-free controls in media with standard pH 6.8 and lower pH 5.6
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were done. While Mtb did not show significant replication in the low pH
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medium, in standard conditions we detected increase in CFU counts after 7
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days of incubation. Therefore drug effects are shown as percentage survival at
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the times indicated with respect to the counts determined in the inoculum.
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Survival percentages (%SR) were calculated separately for days 3 and 7
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using the following formula: %SR=(Viable count treated/Viable count) at 0 time
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point x100. The assay abbreviations, their target populations and their
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interpretation are summarized in Table 1.
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Control experiments were conducted to compare antimicrobial tolerance of
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Mtb from sputum with those of the H37Rv laboratory strain and in vitro
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passaged sputum isolates. H37Rv was grown in supplemented 7H9 medium
8
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to an optical density of 0.8 measured at 580 nm (OD580) and used for drug
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treatment as described above. Sputum isolates were passaged twice in
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supplemented 7H9 medium prior drug tolerance assays. To assess the effect
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of the decontamination procedure and freezing on Mtb cultures, H37Rv and
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the S20 sputum isolate grown in 7H9 medium to an OD580 0f 0.8 were
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subjected to the decontamination procedure and frozen in 10% glycerol as
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described above. In control experiments grown Mtb cultures were spiked in
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non-tuberculosis sputum followed by decontamination and freezing. In all
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experiments Mtb cells were inoculated at ~5x105-1x106 CFU/ml.
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Strain typing and Indirect Drug Susceptibility Testing
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Strains were typed using MIRU-VNTR and their drug susceptibility was tested
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using
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mycobacterial strains were plated on 7H10 agar containing rifampin (1.0 µg/µl)
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isoniazid (0.2 µg/µl) ethambutol (5.0 µg/µl), streptomycin (2.0 µg/µl) and a
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drug free control. Plates were incubated at 37°C checked weekly and at 3
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weeks the number of colonies in the drug containing plates was compared to
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the number of colonies in the drug free plates. No colonies were detected on
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plates containing drugs.
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RESULTS
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Influence of storage medium on cryopreservation of Mtb populations
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from sputum
the
indirect
method
as
described
previously
(15,18)
Briefly,
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Our initial experiments indicated that freezing of non-decontaminated
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samples produced highly variable results between sputum aliquots (data not
205
shown). Therefore, we explored conditions for optimal preservation and
9
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storage of decontaminated homogenized sputum samples. Table S1
207
summarizes data on survival of sputum Mtb in three different solutions: (i)
208
PBS; (ii) 200 mM trehalose in water; (iii) 10% (v/v) glycerol in water (Table
209
S1). Our data show that storage of decontaminated samples in all media
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resulted in significant preservation of colony-forming and SN-dependent
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mycobacteria. In most cases freezing was associated with an increase in
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viable counts (both on agar and in liquid) and the highest counts were
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achieved in 10 % glycerol. These observations suggest that freezing and
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subsequent centrifugation (see Methods) may remove the inhibitory activity
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previously found in sputum in some cases (6). Additional experiments with 5
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separate sputum samples demonstrated that the glycerol solution is a
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satisfactory storage medium for the preservation of Mtb populations in sputum.
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Figure 1 demonstrates that viable counts of mycobacteria recovered from
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sputum samples frozen in glycerol were comparable to or higher than those
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from samples processed before freezing and that the relative amounts
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detected by the three culture methods were preserved. The ability to
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cryopreserve sputum Mtb populations marks an important milestone which
223
enabled us to study drug tolerance patterns in Mtb populations.
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Cidal activity of first line drugs on Mtb populations in sputum
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To investigate the effect of drugs on various Mtb sub-populations, we selected
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high-volume sputum samples from 4 patients. As shown in Figure 2, SN-
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dependent populations dominated all four sputum samples. Sensitivity of the
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isolates from these samples to growth inhibition by all first line agents was
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confirmed by standard susceptibility testing (18) (see Methods). Antimicrobial
10
230
tolerance of the Mtb populations was assessed separately for each sputum
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and the results are summarized in Figures 3 and 4.
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Consistent patterns of efficacy were observed in all tested sputa with respect
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to all agents except rifampin. Therefore the results of the assays based on
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inocula from four sputa are displayed as mean (±SD) % survival (Fig. 3) while
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those for rifampin are displayed separately (Fig. 4). Pyrazinamide showed no
236
effect at 3 days and a modest cidal action at 7 days (