Strain variation amongst clinical and potable water isolates of M. kansasii using automated repetitive unit PCR

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Abstract

Mycobacterium kansasii is a pulmonary pathogen that has been grown readily from municipal water, but rarely isolated from natural waters. A definitive link between water exposure and disease has not been demonstrated and the environmental niche for this organism is poorly understood. Strain typing of clinical isolates has revealed seven subtypes with Type 1 being highly clonal and responsible for most infections worldwide. The prevalence of other subtypes varies geographically. In this study 49 water isolates are compared with 72 patient isolates from the same geographical area (Brisbane, Australia), using automated repetitive unit PCR (Diversilab) and ITS_RFLP. The clonality of the dominant clinical strain type is again demonstrated but with rep-PCR, strain variation within this group is evident comparable with other reported methods. There is significant heterogeneity of water isolates and very few are similar or related to the clinical isolates. This suggests that if water or aerosol transmission is the mode of infection, then point source contamination likely occurs from an alternative environmental source.

Introduction

Mycobacterium kansasii is a pulmonary pathogen found in tap water (Engel et al., 1980a, Engel et al., 1980b). It is the second most common cause of Nontuberculous Mycobacterial (NTM) disease in the United States, South America (Dailloux et al., 2006) and areas of high incidence occur around the world – particularly Poland, Slovakia, Paris, (Hoefsloot et al., 2013) South-east England and Wales. In Queensland, Australia, it is the fourth most common cause of NTM pulmonary disease (behind M. intracellulare, M. avium and M. abscessus) (Thomson, 2010).

The typical presentation of disease is a cavitary upper lobe pulmonary disease, affecting middle-aged men, often with underlying COPD and heavy alcohol intake. Underlying malignancy, HIV co-infection and silicosis are known risk factors for disease. The nodular and bronchiectatic form of pulmonary disease seen with M. avium complex has also been described with M. kansasii. Soft tissue infection can also occur, though is rare. (Evans et al., 1996, Bloch et al., 1998, Maliwan and Zvetina, 2005).

The reservoir of M. kansasii is still not well understood, though it has been postulated that water is the natural habitat. Whilst M. kansasii has been readily isolated from tap water, (McSwiggan and Collins, 1974, Engel et al., 1980b, Steadham, 1980) it has only been rarely isolated from natural water sources such as rivers or lakes. It has been rarely recovered from soil samples or animals. However it is worth noting the technical difficulties in identification of mycobacteria in environmental samples. A definitive link between an environmental source of infection and human disease has not been shown, nor has human-to-human transmission.

Seven subtypes of M. kansasii have been described using amplification of the 16S–23S rRNA spacer region, and PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene (Iinuma et al., 1997, Picardeau et al., 1997) Type I is the most prevalent clinical isolate worldwide (Alcaide et al., 1997, Taillard et al., 2003, Santin et al., 2004). In Queensland typing was performed on 140 stored clinical isolates of M. kansasii. Type I was evident in the small numbers of cases in between 1975 and 1990 and was again the most dominant, with other types seen in the subsequent years as case notifications increased. (Psaltis, 2005, Conference presentation) (Fig. 1).

The aim of this study was to compare M. kansasii strains found in potable water and patient houses to clinical isolates from QLD patients. In Queensland NTM disease remains notifiable and a central reference laboratory performs speciation of all culture positive isolates, providing a unique opportunity to capture patient isolates from the whole state. This paper reports the comparison of these water isolates with human isolates collected from patients in Queensland during the same time period.

Section snippets

Methods

During 2007–2008 citywide water sampling of approximately 220 sites (Trunk Main, Reservoir and Distribution samples) was performed and 49 isolates were identified as M. kansasii using 16s rRNA and hsp 65 gene fragment sequencing. A home sampling study of water, swabs and aerosols from NTM patients houses was also conducted in 2009–2010 and 16 isolates of M. kansasii were recovered. M. kansasii ATCC strain 12478 was included for comparison. Water isolates had been stored in Dubos broth at −20 °C

Results

The mean age of the 68 patients was 62.6 years (range 2–92 years). Forty-one (60.3%) were male. The majority (92.6%) of patients had pulmonary infection, four had soft tissue isolates and one isolate came from a child's cervical lymph node.

Using a similarity cut off of 97%, there were 33 strain types identified amongst clinical isolates (including the ATCC reference strain). Fig. 2 shows the Scatterplot and Fig. 3 the Dendrogram of clinical isolates. Fifty-five of these were identified as Type

Discussion

In Queensland, the incidence of disease due to NTM has been increasing (Thomson, 2010) and between 2007 and 2010, water sampling was conducted to investigate the presence of pathogenic NTM in potable water (Thomson et al., 2013a) and in the homes of NTM patients (Thomson et al., 2013b). M. kansasii was one of the dominant pathogenic species found in these samples, yet M. kansasii disease is not common in Queensland.

In this study we have shown that automated rep-PCR has sufficient discriminatory

Acknowledgements

This study was funded by grants from the Gallipoli Medical Research Foundation of Greenslopes Private Hospital and by The Prince Charles Hospital Foundation. The authors would like to acknowledge the assistance Dr Chris Coulter (QLD Mycobacterial Reference Laboratory) and Dr Hanna Sidjabat (UQCCR).

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