Mycobacterium tuberculosis Uganda II is more susceptible to rifampicin and isoniazid compared to Beijing and Delhi/CAS families

Study site and setting

This was a laboratory-based study conducted at the Uganda National Tuberculosis Reference
Laboratory (NTRL) and the Medical Molecular Laboratory of the Department of Medical
Microbiology, College of Health Sciences, Makerere University. The NTRL is a fully
equipped Biosafety Level 3 Laboratory where TB culture is routinely done. Molecular
tests to confirm Mycobacterium isolates to species level, as well as strain families, were performed at the Medical
Molecular Laboratory.

Isolates and drug susceptibility testing

Mycobacterium tuberculosis isolates were obtained from the Joint Clinical Research Centre (JCRC), Kampala. Drug
susceptibility testing was previously performed at the JCRC and the isolates were
found to be susceptible to both Isoniazid and Rifampicin. The isolates were previously
confirmed to species level through Region of Difference PCR analysis 4], and genotyped by MIRU-VNTR to confirm they were M. tuberculosis Beijing, Uganda II, and Delhi/CAS families 7], 17].

At NTRL, the retrieved isolates (Beijing, Uganda II and CAS/Delhi) were sub-cultured
by incubating at 37 °C for 21 days on Middlebrook 7H11 agar supplemented with OADC.
Bacterial suspensions used in subsequent assays were prepared by resuspending single
colonies of each strain into 1 ml of sterile distilled water.

Drug susceptibility testing was repeated using phenotypic and genotypic approaches;
Middlebrook 7H10 agar proportion method and line probe assays (Genotype MTBDRplus,
Hain Life Sciences, Germany), respectively. With both methods all the isolates were
confirmed to be fully susceptible to Isoniazid and Rifampicin.

Genotyping

Chromosomal DNA used as templates in PCRs was prepared as previously described 17]. Similarly, the M. tuberculosis spoligotypes were determined by Spoligotyping as recently described 6]. The isolates were re-genotyped using MIRU-VNTR 17] and SNP-based Real-Time PCR 8] to confirm that they belong to M. tuberculosis Beijing, Uganda II and Delhi/CAS families.

Determination of minimum inhibitory concentrations

Isoniazid and Rifampicin were purchased from Sigma-Aldrich (St. Louis, MO). Stock
concentrations at 10 mg/ml were prepared using either sterile distilled water (Rifampicin)
or Dimethyl Sulphoxide (DMSO) (Isoniazid). To determine the minimum inhibitory concentrations
(MICs), microdilution plate assays were performed as described by Wallace et al.,
1986 18] and Lenaerts et al., 2005 19]. Bacterial suspensions were adjusted to McFarland standard of 0.5, 100 ?l of which
were inoculated into 11 ml Middlebrook 7H9 medium supplemented with glycerol and OADC,
to give a desired inoculum of 1 x 10
⁵
colony forming units (CFU) per ml.

Microtitre plate wells were seeded with 98 ?l of the bacterial suspension, into which
2 ?l of drug was added. The final concentration of drugs ranged from 0.03–4.0 ?g/ml
(Isoniazid) and 0.12-16 ?g/ml (Rifampicin). Control wells contained only DMSO or water.
The outer wells of the plates were filled with water to avoid evaporation from the
sample wells. The plates were sealed with perforated parafilm and incubated at 37 °C
for 21 days. Plates were observed weekly to monitor changes in growth. Growth inhibition
was determined by visual examination. The MIC was determined as the lowest drug concentration
with no visible bacterial growth after 3 weeks of incubation. Duplicate MICs per strain
were determined. In all assays the M. tuberculosis reference strain, H37Rv, was used as the control.

Determination of killing curves under aerobic conditions

The MICs above were used to calculate the final drug concentration per strain that
was used in determining killing curves. The bacterial suspensions were adjusted to
an inoculum of Log
₁₀
7.0 CFU/ml (range of Log
₁₀
6.9–8.2 CFU/ml), as described above. To the bacterial suspension, drugs were added
to final concentrations of; 1x MIC, 2x MIC and 5x MIC, and incubated at 37 °C. As
such, the concentrations for Isoniazid were: 0.5 ?g/ml (1x MIC), 1 ?g/ml (2x MIC)
and 2.5 ?g/ml (5x MIC) for Uganda II; 0.03 ?g/ml (1x MIC), 0.06 ?g/ml (2x MIC) and
0.15 ?g/ml (5x MIC) for both Beijing and Delhi/CAS. Further, the concentrations for
Rifampicin were: 1 ?g/ml (1x MIC), 2 ?g/ml (2x MIC) and 5 ?g/ml (5x MIC) for Uganda
II; 0.12 ?g/ml (1x MIC), 0.24 ?g/ml (2x MIC) and 0.6 ?g/ml (5x MIC) for both Beijing
and Delhi/CAS.

The day on which the drug was added to the cultures was defined as day 0 and the corresponding
CFUs were taken as 100 %. On days 0, 2, 4, 6, 8 and 10, one milliliter was drawn from
each tube and serially diluted with sterile distilled water. One hundred microliters
of the diluted culture was plated onto Middlebrook 7H11 agar supplemented with OADC,
and incubated at 37 °C under normal atmospheric conditions. After 21 days, CFUs/ml
were determined and were used to calculate the log
₁₀
CFUs/ml.

Determination of killing curves under oxygen depleted conditions

We employed Wayne’s model as described by Murugasu-Oei et al., 2000 20] with a few modifications to suit our setting. Bacterial suspensions were prepared
as explained above. Tubes were closed with sterile silicone rubber septa, and incubated
at 37 °C with slow stirring for 24 days. Control tubes, in addition to the culture,
contained the Methylene Blue dye (1.5 g/ml), which was used as an indicator of oxygen
depletion. The blue dye faded and disappeared indicating that oxygen is successfully
depleted in the culture, as described by Wayne and Hayes, 1996 21]. The drugs were injected into cultures through the septa on day 24 at concentrations
based on MICs. Controls had no drug but water or DMSO. The cultures were incubated
at 37 °C for 10 days.

On days 0, 2, 4, 6, 8 and 10, one milliliter from each tube was serially diluted in
sterile distilled water, and 100 ?l of the diluted culture incubated at 37 °C on Middlebrook
7H11 agar plates supplemented with OADC under normal atmospheric conditions. Colonies
were counted after 21 days.

Data analysis

To test the hypothesis that there is a difference in response to anti-TB drugs between
M. tuberculosis Uganda and other families, data were analyzed with the Student’s t-distribution test
to compare responses of M. tuberculosis Uganda II vs. Beijing, and Uganda II vs. Delhi/CAS. The means of the different killing
curves were compared and a p-value of 0.05 at 95 % CI and ??=?0.05 was considered significant. The data was analyzed
with GraphPad Prism statistical program version 5.

Definitions

In this study, strain refers to any of the seven MTBC lineages 3] or sub-lineages; hence, strain?=?lineage/sub-lineage.