Effect of common and experimental anti-tuberculosis treatments onMycobacterium tuberculosisgrowing as biofilms

Abstract

Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment.

Isolation of a Novel Polyketide from Neodidymelliopsis sp.

“…M. abscessus BSG301 and M. marinum BSG101 [ 15 ] are stable bioluminescent derivatives transformed with the integrating plasmid pMV306G13ABCDE [ 16 ]. This allows light production to be used as a surrogate for bacterial viability [ 15 , 17 , 18 , 19 ]. Mycobacterial cultures were grown with shaking (200 rpm) in Middlebrook 7H9 broth (Fort Richard, Auckland, New Zealand) supplemented with 10% Middlebrook ADC enrichment media (Fort Richard, Auckland, New Zealand), 0.4% glycerol (Sigma-Aldrich, St. Louis, MO, USA), and 0.05% tyloxapol (Sigma-Aldrich, St. Louis, MO, USA).…”

“…Between measurements, plates were covered, placed in a plastic box lined with damp paper towels, and incubated with shaking at 100 rpm at 37 °C for M. abscessus and 28 °C for M. marinum . Bacterial luminescence was measured at regular intervals using a Victor X-3 luminescence plate reader (PerkinElmer, Boston, MA, USA) with an integration time of 1 s. We have defined the MIC as causing a 1 log reduction in light production, as previously described [ 17 , 18 ]. More detailed protocols are available at protocols.io (accessed on 24 May 2021) [ 20 , 21 ].…”

Screening of Fungi for Antimycobacterial Activity Using a Medium-Throughput Bioluminescence-Based Assay

“…Between measurements, plates were covered, placed in a plastic box lined with damp paper towels, and incubated with shaking at 100 rpm at 37 • C for M. abscessus and 28 • C for M. marinum. We measured bacterial luminescence at regular intervals using a Victor X-3 luminescence plate reader (PerkinElmer) with an integration time of 1 s. We have defined the MIC as causing a 1 log reduction in light production, as previously described (Dalton et al, 2016(Dalton et al, , 2017. We have published a more detailed description of our methods on the protocol repository website protocols.io (Wiles and Grey, 2021c,d).…”

Biofilm Formation Induced by Clinical Isolates of Mycobacterium Tuberculosis

“…Одним из наиболее значимых свойств возбудителя внутри БФ является резкое снижение его чувствительности к воздействию антибиотиков. МБТ не являются исключением: возбудитель внутри БФ в 20-1000 раз более устойчив к противотуберкулезным препаратам (ПТП) [6]. Однако продукция БФ клиничес кими штаммами туберкулеза, остается весьма загадочным феноменом.…”

“…Анализ более трех сотен клинических штаммов из двух областных противотуберкулезных учреждений Иркутска и Улан-Уде [3] показал, что в подавляющем большинстве случаев клинические изоляты МБТ не имеют способности к продукции БФ. Анализ литературы свидетельствует, что ни в одном из исследований за последние десятилетия нет описания масштабной продукции БФ клиническим штаммами, и все описанные эксперименты проведены на лабораторных штаммах, зачастую подвергнутых генной модификации [6,7,11,16]. В то же время в русскоязычной литературе 80-х гг.…”