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BCCM/ITM Mycobacteria Collection

 

BCCM/ITM has been operational since the 1st of February 2011, and harbours one the largest and most diverse collections of well-documented mycobacteria worldwide, including the TDR TB-Strain bank.

Embedding

BCCM/ITM is hosted by and sharing its research interests with the Mycobacteriology Unit at the Institute of Tropical Medicine in Antwerp, dedicated in research to combat tuberculosis, leprosy, Buruli ulcer and other mycobacterial diseases. Research activities benefit from the leading role of the unit as Supranational Reference Laboratory for tuberculosis, providing technical assistance and services to clinical laboratories at national and international level

Building on the strong expertise in multiple areas of mycobacteriology, the unit has initiated new approaches to better understand:

  • Mechanisms underlying resistance to old (rifampicin, isoniazid) and newer (fluoroquinolones, bedaquiline) drugs
  • Transmission dynamics of M. tuberculosis and M. leprae
  • Phylogeography of M. tuberculosis complex
  • Phenotypic and genotypic differences among members of the M. tuberculosis complex
  • Phylogeny of non-tuberculous mycobacteria
  • Optimization of biosafety containment around working with resistant M. tuberculosis.

 

History of the collection

 

Scope of the biological material

BCCM/ITM is a collection of well documented mycobacteria, characterized by phenotypic and/or genotypic tests. It comprises strains from human, animal and environmental origin from all continents spanning from 1960 to date, covering more than 90 mycobacterial species.

While having an emphasis on (drug-resistant) M. tuberculosis complex, all mycobacterial species can be deposited in the BCCM/ITM collection.

BCCM/ITM greatly welcomes deposits of Type strains for newly described species, and accepts pathogens up to risk class 3.

Major publicly available sub-collections include (but are not limited to):

  • M. tuberculosis clinical isolates from the WHO-TDR TB-Strain Bank (Vincent et al., 2012).
  • The ‘clinical reference set’ of M. tuberculosis strains comprising lineages 1 to 7 (L1-L7) (Borell et al., 2019)
  • The M. tuberculosis Lineage 8 strain, the only known viable isolate worldwide (Ngabonziza et al., 2020)
  • In vitro-selected drug-resistant M. tuberculosis strains (clofazimine, bedaquiline, linezolid, fluoroquinolones, second-line injectables, ….)
  • M. tuberculosis complex strains from animal origin (M. bovis, M. orygis, M. pinipedii, M. caprae, M. microti, …)
  • Type strains from non-tuberculous mycobacteria (e.g. Fedrizzi et al., 2017)
  • Two large transposon insertion mutant libraries (96X96 wells) of M. bovis BCG

 

Mycobacterial species information

An overview of the current species names in the genus Mycobacterium can be consulted here (last update September 2018). This overview includes the species names and supplementary info that can be useful for research purposes.

More detailed information on the clinical relevance of non-tuberculous mycobacteria (NTM) can be consulted here (update January 2020).

 

Quality

Accession, control, preservation, storage and supply of mycobacteria and related information in the frame of public deposits are ISO 9001:2015 certified. The control of viability, identity and mycobacterial purity on the batches for release, are performed according to the ISO 15189 standard (BELAC 147MED).

 

Phenotypic drug-susceptibilty testing for Mycobacterium tuberculosis complex (MTBc) isolates at BCCM/ITM

To determine the drug-susceptibility profile of MTBc isolates, the proportion method is the most commonly applied (Rigouts & Cogneau, 2021).

It establishes the proportion of bacilli growing on culture-containing medium as compared to drugfree slants, considering an isolate as clinically resistant if >1% of the bacterial population resists the drug. To this end, an appropriate bacterial suspension (e.g., 10−2 or 10−1 dilution MacFarland #1) of the isolated culture is inoculated on the drug-containing slants, while a 1:100 dilution of the same suspension is inoculated on ad drug-free control.

Requirements for the analyte: Viable pure mycobacterial culture isolates on solid or in liquid medium. Upon arrival we will subculture the MTBc on Löwenstein-Jensen (LJ) medium or in liquid MGIT medium to initiate the desired testing:

  1. proportion method
    1. on solid medium (LJ and/or Middlebrook 7H11 agar medium)
    2. in liquid medium (MGIT960)
  2. determination of the minimal inhibitory concentration (MIC)
    1. on solid medium (LJ and/or Middlebrook 7H11 agar medium)
    2. in liquid medium (MGIT960)
    3. using the microdilution broth method (EUCAST standard) (ref)

Expected turn around time:  Solid medium testing may take up to 3 months after arrival, while liquid medium testing may be available in 2 months. In case > 10 strains need to be tested, this may however take more time.

 

References: Rigouts & Cogneau, 2021 eBook ISBN9781003099277

 

Phenotypic identification at BCCM/ITM

Phenotypic identification of mycobacteria is based on a set of characteristics, such as resistance to certain growth inhibitors, biochemical reactions, colony morphology, the ammonium ferric citrate test, and the urease, catalase and Tween hydrolysis tests. Final identification of a species is determined by the combination of results from various tests.

 

The following phenotypic assays are standard available at BCCM/ITM

  • Temperature inhibition: This test examines whether bacteria can grow in 37°C as compared to 30°C. With the exception of Mycobacterium ulcerans, for instance, most non-pathogenic mycobacteria that are isolated from the environment are not resistant to these higher temperatures.
  • Pigment formation: This test examines whether bacteria are capable of forming pigment with or without prior exposure to sunlight. For this test, it is important to use well-grown, fresh and well-ventilated cultures. The kind of solid medium itself is of little importance.
  • Colony morphology: Through the growth on an enriched agar plate, both the colony morphology and the purity of the culture are examined. This also allows for a simple colony subculture to be created in the event of partial contamination.
  • MPT64 antigen test: This rapid, lateral flow assay differentiates species from the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria.

The below presented phenotypic assays can be performed upon specific request BCCM/ITM

  • Inhibitor series: Here, the extent to which the growth of mycobacteria is inhibited by the presence of certain products (inhibitors) is investigated. This growth is compared to growth in a tube with 1/100 bacterial dilution, i.e. 1% of maximum potential growth.
  • Urease test: This test examines whether bacteria have an active urease-enzyme; in other words, whether they can break down urea in this medium. Through the breakdown of urea, ammonium forms, which, in turn, raises the pH and causes the neutral red indicator to colour the medium pink.
  • Tween hydrolysis test: This test examines whether bacteria can breakdown the Tween present in the medium. Through the breakdown of Tween, pH rises, which in turn makes the neutral red indicator change the medium from orange to red.
  • Catalase test: This, actually, refers to the semi-quantitative catalase test. This test examines whether the bacteria possess the catalase enzyme that can convert hydrogen peroxide into water and oxygen (2H202 → 2H2O + O2). The released oxygen generates the foam in the mixture containing detergent (Tween). The amount of foam produced is measured.
  • Niacin production test: This test examines whether bacteria produce niacin in the medium. Nicotinic acid (niacin) plays a vital role in oxide-reduction reactions that occur during the metabolic processes of mycobacteria. Although all mycobacteria produce niacin, it has been shown that, through a blocked metabolic reaction, M. tuberculosis exhibits by far the largest accumulation of niacin, making it a commonly used identification test. The production of media containing egg generates the best niacin production and is recommended when performing this test. The culture must also be at least three weeks old and contain at least 50 colonies. To optimally free the produced niacin from the medium, it is advisable to remove the pre-grown colonies beforehand, especially in cases of fully-grown cultures. Ventilating the culture beforehand is also essential for the success of the test.
  • Nitratase Test: This test examines whether bacteria can reduce nitrate. The culture to be tested should be fresh and contain at least 50 colonies. This is achieved by placing the bacterial suspension in contact with a nitrate solution for a minimum of 2 hours, after which the nitrate content is measured by adding sulphanilamide and α-napthylamine.
  • Phosphatase test: This test examines whether bacteria can reduce phosphate. The culture to be tested should be fresh and contain at least 50 colonies. This is achieved by placing the bacterial suspension in contact with a di-phosphate solution for at least 2 hours, after which the phosphate content is measured by adding sodium carbonate.
  • Ammonium ferric citrate test: This test examines both the growth rate and the capacity to absorb iron. A very small amount of bacteria is inoculated onto a peptone agar medium. Only fast-growing mycobacteria are able to grow on the poor peptone agar medium, and only a portion of them is able to absorb the iron into their cells. Direct contact of the inoculum with the ammonium ferric citrate solution must be avoided.
  • Arylsulfatase: This test looks at the ability of the bacteria to produce sufficient arylsulfatase. The enzyme arylsulfate will convert phenolphthalein disulfate to free phenolphthalein, which will give the medium a pink to red color. The test can be read after 3 days (for rapid and slow growers) and after 14 days (slow growers).

Requirements for the analyte: Viable pure mycobacterial culture isolates on solid or in liquid medium.

Expected turn around time:  Phenotypic identification may take up to 3 months after arrival, depending on the species (rapid or slow growing) and tests requested.

 

References: Rigouts & Cogneau, 2021 eBook ISBN9781003099277;  Bhalla et al., 2018  doi: 10.1016/j.plabm.2018.e00107;

 

 

Whole genome sequencing

BCCM/ITM does not have the facility to perform whole genome sequencing in house. Following gDNA extraction, we outsource this analysis to an external provider with whom we collaborate for our research projects. The analysis of the generated sequences, can be peformed by BCCM/ITM.

Genomic DNA extracton at BCCM/ITM

To extract genomic DNA (gDNA) from pure mycobacterial culture isolates, we apply an in-house developed method based on the Maxwell extraction (Promega, USA) (Lempens et al., 2018).

Requirements for the analyte: Viable pure mycobacterial culture isolates on solid or in liquid medium. Upon arrival we will subculture the mycobacteria on Löwenstein-Jensen medium or another solid medium meeting the species-specific requirements.

Expected turn around time:  depending on the species’ growth speed, we expect gDNA to be extracted in 4 to 8 weeks. In case > 10 strains need to be tested, this may however take more time.

 

References: Lempens et al., 2018 DOI: 10.1038/s41598-018-21378-x

 

 

Genotypic typing of Mycobacterium tuberculosis complex isolates by spoligotyping at BCCM/ITM

Spoligotyping is a PCR-based method to simultaneously detect and type M. tuberculosis complex (MTBc) bacteria. It is based on DNA polymorphism present at one particular chromosomal locus, the "Direct Repeat" (DR) region, which is uniquely present in MTBc bacteria (Kamerbeek et al., 1997). By spoligotyping we can detect the presence or absence of 43 inter-repeat spacers with known sequences.  

Requirements for the analyte: BCCM/ITM can perform these analysis on pure mycobacterial culture isolates, which can be sent as viable cultures or DNA extracts (heat-inactivated thermolysates or purified genomic DNA).

Expected turn around time:  Depending on the date of receipt and the number of samples to be tested, it can take from 1 to 5 weeks (spoligotyping is done at least every last week of the month, or when sufficient samples are available to fill a complete membrane).

 

References: Kamerbeek et al., 1997 DOI: 10.1128/jcm.35.4.907-914.1997

Genotypic identification by Sanger sequencing of the 16SrRNA and/or rpoB genes at BCCM/ITM

16S rDNA sequence analysis is a standard method in bacterial taxonomy and identification, and is based on the detection of sequence differences (polymorphisms) in the hypervariable regions of the 16S rRNA gene which is present in all bacteria. An unknown isolate can be identified by comparing the similarity of its 16S rDNA sequence with 16S rDNA sequences of strains with known taxonomic identity that are contained in public databases. However, results of similarity searches may be compromised by the quality of publicly available sequences (e.g. NCBI), which are often not validated or guaranteed, and/or the fact that many sequences from uncultured or unnamed organisms are present. Hence, we will always consider comparator sequences from Type strains or reference strains.

To identify mycobacteria to the (sub-)species level we perform a conventional PCR followed by Sanger sequencing (outsourced to BaseClear) using in-house developed primers to target the 16 rRNA gene. In case this approach does not allow to distangle the (sub-)species sufficiently, we perform a second PCR and Sanger sequencing step targeting the hypervariable part of the rpoB gene, using in-house developed primers (Rigouts & Cogneau, 2021). For some species, such as M. ulcerans or M. leprae, we apply a species-specific qPCR, using in-house developed primers targeting IS2404 (WHO, 2014) or RLEP (Braet et al., 2018).

Requirements for the analyte: BCCM/ITM can perform these analysis on pure mycobacterial culture isolates, which can be sent as viable cultures or DNA extracts (heat-inactivated thermolysates or purified genomic DNA).

Expected turn around time: Results will be available in 2 weeks, in case of a single sequence analysis, and may take another week in case additional testing/sequencing is required. In case > 40 strains need to be tested, this may however take more time.

 

The Mycobacteriology Unit hosting BCCM/ITM can perform this analysis directly on clinical specimens (https://labo.itg.be/analysen/detectie-van-mycobacterien-en-m-tuberculosis-complex-door-pcr/ )

 

References: Rigouts & Cogneau, 2021 eBook ISBN9781003099277; WHO 2014, ISBN 978 92 4 150570 3;  Braet et al., 2018 doi: 10.1128/JCM.01924-17

 

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