Tuberculosis- International Perspectives on Epidemiology, diagnosis and Controls
finalseminar_mkansasii_20151106
1. Presentation title
Presenter’s name
Department/Unit/Ward
Service line
Facility/hospital
Date
Molecular analysis of Mycobacterium
kansasii from human and potable
water specimens
Carla Tolson
Queensland Mycobacterium Reference Laboratory
Pathology Queensland
Royal Brisbane and Women’s Hospital, Herston, Qld
Friday 6 November 2015
3. Introduction – What are Mycobacteria?
•Mycobacteria are aerobic, gram-positive, acid-fast
organisms
•Mycobacteria are acid-fast due to high lipid content in
the cell wall
•Currently 172 species, most recognised is Tuberculosis
Mycobacterium species
M. tuberculosis complex Non-tuberculous
(Pathogenic) mycobacterium (NTM)
(non-pathogenic)
4. •Predominantly pulmonary infections, can cause disease
in other areas of the body
•Immuno-compromised patients, e.g. COPD, HIV, etc
•Diagnosis based on symptoms, radiology, microbiology
•Incidence of significant pulmonary disease is increasing
•The most common NTM isolated in Queensland are:
– M. intracellulare
– M. avium
– M. abscessus
– M. fortuitum
– M. kansasii
Introduction – How do NTM’s affect humans
5. • M. kansasii is a pulmonary pathogen, readily grown from
tap water, rarely from natural waters
• A definitive link between water exposure and disease
has not been proven
• Environmental niche poorly understood
• Typically cavitary lung disease, resembling TB
← Normal CXR
Abnormal CXR →
Introduction – M. kansasii
6. Introduction – M. tuberculosis vs. M. kansasii
Macroscopic
Microscopic (x100)
M. tuberculosis complex M. kansasii
7. Introduction – M. tuberculosis vs. M. kansasii
• Immunochromograpic test
• Molecular detection
8. Why is genotyping important?
• Assist with identification of patient to patient transmission
• To link environmental sources with human disease
9. Background – Genotyping M. kansasii
• Typing of M. kansasii was first published in 1975 by
Engel using phages
• Very few publications on strain typing until 1997
• A range of DNA based methods have been used include
– hsp65 PCR-Restriction Fragment Length
Polymorphism / hsp65 PCR-restriction enzyme analysis
– AccuProbe Gen-Probe identification
– PFGE of the 16S-23S intergenic spacer region
– Large restriction fragment analysis
– Amplified Fragment Length Polymorphism
– 16S-23S ITS region / Restriction enzyme analysis
10. Environmental M. kansasii
• Clusters associated with mining and industrial zones
– Gold Mine in South Africa
– Coal & Silica Mines in Netherlands
– Metal/Welding in Europe
11. Worldwide strain type distribution of M. kansasii
Europe (incl: SPA, FRA, NED, BEL)
type I,II,III,IV,V,VI Human &
Environment
USA - type I,II,III from
human only
Africa - unknown
Japan - type I,II,III,IV,V
Human & Environment
15. Knowledge Gaps
• How many strains exist in the environment?
• Could there be many more out there?
• Are these strains present (particularly type I) only in
humans or are they found in the environment as well?
• Can newer methods characterise subtypes better?
16. Hypothesis
•That newly developed DNA-based genotyping methods
are able to characterise and differentiate clinically and
environmentally sourced strains of Mycobacterium kansasii
17. Aims
• Aim 1 – To genotype all M. kansasii isolates using new
and existing genotyping methods
• Aim 2 – To compare the genotypes of M. kansasii
isolates from clinical samples to those obtained from
Brisbane water and patient home water samples
21. repetitive DNA
elements distributed
more or less
randomly over the
genome.
primers that anneal to these repetitive elements
Separation and detection
of products by
electrophoresis using
micro-fluidic chips
DNA Fingerprints analysed with Diversilab®
software-Pearson correlation co-efficient and
unweighted pair group method with arithmetic
means to compare isolates and determine
clonal relationship.
Method 1 – DiversiLab automated rep-PCR
22. Method 2 – High Resolution Melt Analysis (HRM)
• PCR is performed to specific Target
tagged with a fluorescent dye
• HRM is run immediately following
• dsDNA to ssDNA melt is observed
24. Results – HRM
• Rapidly determined isolates to be “same” or “different” to
M. kansasii ATCC12478 strain
• Currently no publications using HRM for M. kansasii,
validation required
• Validation done by performing ClustalW Analysis on the
16S rRNA sequences to visualise the SNP’s and from
there a Dendrogram was created using Geneous
30. Results – HRM
Dendrogram of
16S rRNA
sequences using
Geneious software
Strain type V
Strain type IV
Strain type I
31. Results – DiversiLab
• Gave the best strain differentiation giving 31 different
fingerprint clusters
• Easy to use software for analysis
• Achieving adequate DNA concentration for this
assay was a challenge – repeat runs
33. Results – DiversiLab
• 2 isolates (WP5) matched
the dominant clinical strain
type CP11.
• A dominant cluster of
water isolates (WP2)
matched a single patient
isolate (CP9).
• The remaining water and
clinical isolates were
unrelated.
ATCC Strain
Type I – dominant cluster
Type IV
Type V
34. Results – Summary of HRM vs. DiversiLab
• Both methods demonstrated the main strain types of
M. kansasii
• HRM with the validation data from dendrogram
matched previous strain types defined by ITS-REA
• DiversiLab demonstrated ITS-REA groups with better
strain type differentiation compared to HRM
35. Results – Summary
Method ITS-REA DiversiLab HRM
Number of types
produced
3 31 Same or Different
Typeability 100%
80-100% (after
re-extraction)
95-100%
Speed (TAT) 2 days 5 hours 4 hours
Cost (per isolate) $30~ $48~ 50 cents~
Skill level
required
Basic-
Intermediate
Intermediate
Basic-
intermediate
36. Conclusions
• We were able to successfully type both human and
environmental strains of M. kansasii
• ITS type I, IV & V strains were found among the QLD patients
and Brisbane Water isolates
• Newer typing methods delivered better strain differentiation
than ITS REA, however both have limitations
• Municipal water in Brisbane unlikely to be the source of
infection for patients with M. kansasii disease
• However, can’t exclude water contamination in other areas
associated with mining and industry
37. Discussion
• DNA-based assays are considered the gold standard
• DNA was difficult to obtain using prescribed kit
• DiversiLab demonstrated the best strain differentiation
• Water isolates have a different strain profile to patient
isolates.
• Worldwide, our patterns are different ?Area specific
38. Future Directions
• Whole Genome Sequencing!
• Acquire supplementary reference strains to further
validate HRM
• More sampling from patients houses outside of the
Brisbane Metropolitan with M. kansasii infections
– In particular Western Queensland
39. Future Directions
• Roma- first site of gas discovery 1906; open cut coal mining
• First Report on the Coal Industry of Queensland (1949) “What is striking
is the assessment of coal resources by discoveries of coal found in
water bores. There is water in coal, a lot of it, and there is gas too.”
40. References
-Thomson RM. Changing epidemiology of pulmonary nontuberculous mycobacteria infections. Emerg
Infect Dis 2010 October; 16(10): 1576–1583.
-Respiratory Medicine, Volume 98, Issue 8, August 2004, Pages 721–725
http://dx.doi.org/10.1016/j.rmed.2004.02.011.
-Psaltis, J., Mycobacterium kansasii: A Queensland Mycobacterium Reference Laboratory Review
1975-2004., in Australian Society of Microbiology Annual Scientific Meeting2005: Canberra, Australia.
-Engel HBL, Havelaar, AH. The occurrenece of Mycobacterium kansasii in tapwater. Tubercle
1980;61:21-26.
-McSwigganDA CC. The isolation of M.kansasii and M.xenopi from water systems. Tubercle
1974;55:291-7.
-R. Santos F. Oliveira JF, et al. Detection and identification of mycobacteria in the Lisbon water
distribution system. Water Science & Technology 2005;52(8):177-180.
-Wright E, Collins C, et al. Mycobacterium xenopi and Mycobacterium kansasii in a hospital water
supply. Journal of Hospital Infection 1985;6:175-8.
-Vaerewijck MJ, Huys G, et al. Mycobacteria in drinking water distribution systems: ecology and
significance for human health FEMS Microbiology Reviews 2005;29(5):911-934.
-Roth A, Reischl U, et al. Novel diagnostic algorithm for identification of mycobacteria using genus-
specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases Journal of
Clinical Microbiology 2000 Mar;38(3):1094-104.
41. Acknowledgements
• Supervisors:
–Assoc. Prof. Flavia Huygens (QUT)
–Dr Rachel Thomson (GMRF, UQ, Queensland Health)
–Dr Irani Rathnayake (QUT)
• Colleagues at QMRL
• Study, Education and Research Trust Fund (SERTF/SERC)
• Gallipoli Medical Research Foundation
As a brief overview, what will be discussed today is:
An introduction about mycobacterium
Background into strain typing and why it is important in a clinical setting
The hypothesis
The main aims and objectives to this research
Methods used to strain type M. kansasii
A summary of results obtained
A brief discussion of the results
Conclusions and
The future directions of where this research could lead onto
Mycobacteria are bacteria that are grown aerobically, gram positive and are acid-fast staining
Mycobacteria are distinguished from other bacteria by their characteristic thick cell wall. The cell wall is hydrophobic and rich in mycolic acids which are unique to Mycobacteria. This helps in retaining its staining properties, as well as their longevity to survive.
Currently there are 172 species of Mycobacterium, and the most recognised species is tuberculosis.
Mycobacterium can be separated into two predominant groups, these are M. tuberculosis complex which are pathogenic and Non-tuberculous mycobacterium which are considered non-pathogenic but recent publications suggest transmission between patients for some species.
NTM’s are predominantly pulmonary infections but they can also cause disease in other parts of the human body
NTM are opportunistic infections which tend to affect immuno-compromised patients
Diagnosis of NTM disease is based on symptoms, the radiological appearance of scans in correlation with microbiology
The incidence of significant NTM pulmonary disease is increasing in Australia and world-wide due to the ageing population
Specifically looking at QLD, the most common isolated NTM pathogens are M. intra, M. avium, M. abscessus, M. fortuitum & M. kansasii
Focusing in now to M. kansasii
Mycobacterium kansasii is largely a pulmonary pathogen but has been known to cause disseminated disease
It is readily grown from tap water but rarely from natural waters such as streams and rivers
A conclusive relationship between water exposure and infection has not been proven
The environmental niche of M. kansasii is still poorly understood
Typically the radiological appearance is CAVITREE lung disease, which is similar to TB
As kansasii disease can clinically resemble TB, it is important to differentiate between the two species and from a public health perspective.
In the laboratory, a variety of methods are used to identify between the both such as:
The macroscopic & microscope appearance of the organism
but mostly we use molecular methodologies as well as rapid immunochromographic tests
Genotyping is an important tool for establishing the epidemiology of outbreaks of infection
It can assist with the identification of patient to patient transmission or link environmental sources to human disease
Briefly in the literature, strain typing of M. kansasii was first published in 1975 by Engel by using bacteriophages.
Very few journals articles were published between 1975 and 1997 on typing of M. kansasii
On the discovery of PCR in the 1980’s, many DNA based methods for typing were invented
Previously published methods to type M. kansasii include:
A previously published article suggested that the cause of a cluster of M. kansasii patients with the disease was a gold mine in South Africa.
Another article described coal mines and silica could have been the source for the increase incidence of M. kansasii amongst its workers in Netherlands
From the literature, the following strain types have been demonstrated:
From a study performed in 2004 at QMRL – all M. kansasii isolates identified from 1975-2004 were strain typed using the method published by Roth et al in 2000
The research conducted revealed most of the strains isolated to be type 1
This correlates with publications worldwide indicating that type 1 is the most prevalent
Roth et al 2000 published a robust typing method to not only strain type M. kansasii but also differentiate to a species level other mycobacteria
The figure above is the key developed and published
As this method was used previously to type Queensland strains, we also used the same method to type our isolates. This was important to do as we could then compare the strain types we identified from this study to the previous one
This method was also used as our baseline to validate and identify the strain types from the newer methods
ITS-REA is initially a PCR reaction using specific primers for the ITS region. Using the amplicon generated from the PCR, an restriction enzyme digest is performed using 3 specific targets which cuts at specific points in the DNA strand, to generate a profile. This is then visualised on agarose gel electrophoresis
A specific pattern was created by the enzyme digest and was visually inspected and assigned a individual strain number
Currently six strain types are accepted amongst researchers but there was a seventh identified by Taillard et al. in 2003
From the hypothesis we conceived 2 main aims. Within the two aims further objectives were devised
We firstly captured all M. kansasii patients from 2005-2010 as well as the M. kansasii isolates from the Brisbane Water Study (61) conducted by Dr Rachel Thomson in 2007 & 2008.
Including the ATCC strain available, the total isolates was 140
The isolates were recovered from frozen storage an inoculated on LJ slopes until sufficient growth was available for DNA extraction
Using the UltraClean Microbial DNA extraction (MoBio) kit, all isolates were extracted using the specific Mycobacteria protocol and quantified by spectrophotometry
DNA concentrations were noted
The DiversiLab system is an Automated platform that uses repetitive PCR technology to provide a DNA fingerprint for complete isolate characterisation. Briefly, a PCR mixture was prepared according to the manufacturer’s instructions and Rep-PCR products were separated and detected by using microfluidic chips of the DiversiLab system. DNA fingerprints……..
HRM analysis is used to characterise DNA samples according to their dissociation behaviour as they transition from double stranded DNA to single stranded DNA with increasing temperature. The target sequence must first be purified to high copy number prior to HRM. This is done by a PCR procedure using the RotorGene6000 in the presence of a dsDNA intercalating fluorescent dye like SYBRGreen. The dye does not interact with ssDNA but actively interacts with dsDNA and fluoresces brightly. Initially, fluorescence is high because the sample starts as dsDNA, but fluorescence diminishes as the temperature is raised and DNA dissociates into single strands. The observed melt is then visualised for each sample.
From the results obtained from our baseline assay ITS-REA, a high amount of patient isolates were type 1 and very few type 4’s & 5’s
The environmental isolates were the opposite with more type 4’s & 5’s and only one type 1
HRM demonstrated itself as a highly sensitive PCR assay. With confidence we could determine whether the isolate was considered the same as the type strain or different. Determining the type for each isolate used a value of +/- 5 Units against the type strain as previously described by Price et al & Stephens et al. As this was the first time using HRM to strain type M. kansasii, further validation was needed
16S sequencing was performed on all isolates and using BioEdit software, a ClustalW analysis of the isolates was prepared to visualize the single nucleotide polymorphisms from the type strain. Further validation using this sequencing data was performed by producing a dendrogram using the Geneous software
This is a graphical representation of same vs different numbers
Only 15 patients were the same as the type and 63 were different
No water isolates matched the type strain, which is not a surprise considering there was only one type 1 found
The software from Qiagen created the normalized graph above.
This is showing the characteristic melt of the double stranded DNA to single DNA
Because mycobacteria have a high G-C content, the melt temperature is higher than most bacteria
Mycobacteria melt temperature was between 83-85deg Celsius
We normalise the melt curve to demonstrate where the DNA dissociation occurs
To analyze the data, the difference graphs were used. Any isolate less than 5 units were considered the same
and isolates greater than 5 were different.
This is a demonstration of the BioEdit software after clustalW analysis is applied
The ATCC control strain is used as the reference
The 16S is approx 500bp long with most isolates were 2-3 SNP difference from the control strain
From the 16S sequencing the dendrogram above was created using the software Geneious
The following parametes were applied when analysing:
Select Mr Bayers, then Substitution Model JC69
Rate Variation is Gamma and
all other settings are left on Default.
The validation data corresponded with the HRM data produced
As you can see here, 3 branches are evident, supporting the 3 types we found from ITS-REA
The DNA fingerprint generated was grouped by the software automatically
As per the protocol, analysis was performed using the Pearson correlation co-efficient to compare isolates and determine clonal relationships. Any isolates that were 97% similarity were considered to be indistinguishable.
Isolates that showed 95% similarity were considered similar and
isolates with ≤95% were considered to be unrelated.
Because of the bigger numbers- its easier to demonstrate the similarity between strains using a scatterplot – in which each square on the graph represents 5% similarity.
The Brown isolates represent those from potable water, the Green are home sampling isolates and the Pink is from a shower aerosol. The rest are clinical isolates
There were 14 unique strain types amongst the clinical isolates with the majority being the highly clonal strain which correlated with Type 1 ITS_REA pattern
2 water isolates matched this dominant clinical type. The water isolates formed 2 dominant clusters- one of which matched a single patient isolate.
The remaining water and clinical isolates were unrelated.
Strain typing has progressed over time as highly-advanced technologies have become available to better differentiate and subtype species of Mycobacteria. Currently, DNA-based assays are considered the gold standard as the demand for more rapid and cost effective methods increases.
From our study, the optimisation of the DNA extraction was important. We had issues with acquiring a high enough concentration so modifications to overcome this were necessary. Only one kit was valid for all three tests.
DiversiLab demonstrated the best strain differentiation with the method grouping 31 different sub types
ITS-REA showed only 3 strain types present. One less strain type than the previous results found
ITS-REA was a time consuming assay to perform and it was hard to assign the strain type if the gel didn’t develop enough.
HRM worked well but can only be used as a present or absent situation
Qld’s profile of strain types showed to be different than the other predominant types worldwide
The environmental isolates showed a different percentage pattern of strain types seen.
So where to from here?
Acquiring supplementary reference strains to further validate the HRM would be useful and could therefore be implemented into a diagnostic laboratory
Further sampling from patients houses outside of Brisbane Metropolitan with M. kansasii infections in particular Western Queensland
There are several potential explanations for the significantly higher relative incidence of M. kansasii in the Yuleba and Roma areas. Yuleba is a small town and the site of a major processing facility for silica deposits. Silicosis has been identified as a host susceptibility factor for M. kansasii. Moreover, mining activity is common in the Maranoa-Balonne region. Here on the right you can see a map of the oil and gas facilities in QLD overlying the Surat basin. This region is well known for its agricultural and mining activities with a number of developed petroleum and coal seam gas wells. In addition, the water supply for many communities in the region is primarily from private and communal bores, aquifers and rainwater tanks.
