Clinical Microbiology: Open Access

Genotyping of Soil and Clinical Isolates of Burkholderia Species in Myanmar

Nay Myo Aung^1,3*, Khine Khine Su³, Narisara Chantratita² and Chanwit Tribuddharat^{1 1}Department of Microbiology, Mahidol University, Bangkok, Thailand
²Department of Microbiology and Immunology, Mahidol University, Bangkok, Thailand
³Department of Microbiology, Defense Services Medical Academy, Yangon, Myanmar
^*Correspondence: Nay Myo Aung, Department of Microbiology, Mahidol University, Bangkok, Thailand, Email:

Received: 09-Feb-2024, Manuscript No. CMO-24-24885; Editor assigned: 12-Feb-2024, Pre QC No. CMO-24-24885 (PQ); Reviewed: 26-Feb-2024, QC No. CMO-24-24885; Revised: 03-Apr-2025, Manuscript No. CMO-24-24885 (R); Published: 11-Apr-2025, DOI: 10.35248/2327-5073.25.14.429

Introduction

Burkholderia species are found in various environments, including soil, water and plants [1]. These bacteria are capable of causing severe diseases in humans, animals and plants and their clinical importance is well-recognized. Burkholderia species have been implicated in various infections, including pneumonia, urinary tract infections, sepsis and wound infections [2]. In particular, B. pseudomallei area significant cause of morbidity and mortality in regions where it is endemic, including Southeast Asia and northern Australia [3].

Burkholderia species exhibit high genomic diversity, making molecular genotyping crucial for their identification and classification [4]. Molecular genotyping methods have been shown to have higher resolution and discriminatory power than traditional phenotypic methods, making them a more reliable tool for identifying and classifying Burkholderia species [5]. Two commonly used molecular genotyping methods for Burkholderia species are recA and 16S rRNA gene sequencing.

The recA gene encodes a recombinase enzyme involved in DNA repair and recombination. It is highly conserved among bacterial species and has been used as a target for molecular genotyping and phylogenetic analysis of Burkholderia species [6]. In contrast, the 16S rRNA gene is highly conserved in all bacterial species and is involved in translating mRNA to protein. It has been widely used as a target for molecular identification and classification of bacterial species, including Burkholderia species [7].

Several studies have compared the usefulness of recA and 16S rRNA sequencing for identifying and classifying Burkholderia species. One study reported that recA sequencing had a higher discriminatory power and was more effective in identifying B. pseudomallei isolates than 16S rRNA sequencing [8,9]. Another study found that recA sequencing could differentiate closely related Burkholderia species, while 16S rRNA sequencing showed limited discriminatory power [10].

In addition to recA and 16S rRNA sequencing, other molecular genotyping methods have been used to identify and classify Burkholderia species. These methods include Multilocus Sequence Typing (MLST), which targets several housekeeping genes and Whole-Genome Sequencing (WGS), which provides a high-resolution view of the genetic diversity of Burkholderia species [11].

In Myanmar, limited information is available on the prevalence, diversity and clinical significance of Burkholderia species. A previous study reported the isolation of B. pseudomallei from soil and water samples in Myanmar [12,13]. However, there is a need to expand the knowledge of the prevalence of other Burkholderia species in the country and to develop cost-effective and reliable molecular genotyping methods for their identification and classification according to local facilities in Myanmar [14].

Molecular genotyping methods are essential for accurately identifying and classifying Burkholderia species. However, rapid screening of molecular genotyping among Burkholderia species in Myanmar was found to be a limitation and low awareness. The current diagnostic methods in Myanmar for identifying Burkholderia species are limited to phenotypic methods, which have limitations in their sensitivity and specificity. Furthermore, more than 16S rRNA sequencing alone to identify Burkholderia species may be required due to the high diversity of Burkholderia species. The development of cost-effective and reliable molecular genotyping methods can contribute to the prevention and control of infections caused by these medically necessary pathogens in Myanmar.

Several molecular genotyping methods have been used to identify and classify Burkholderia species in different settings. WGS has also been increasingly used for identifying and classifying Burkholderia species, providing a high-resolution view of the genetic diversity and evolution of these organisms [15,16]. However, these methods can be resource-intensive and costly, limiting their use in resource-limited settings such as Myanmar. This study highlighted the simple discrimination of Burkholderia species among clinical and soil isolates by using a local affordable and properly facilitated laboratory in Myanmar.

Materials and Methods

Bacterial strain collection

Sixteen clinical isolates of Burkholderia pseudomallei were taken and assessed for 2 sequencing assays in a previous study [17]. We collected 29 soil isolates from a gift of the study of Htun et al. [13]. At first, those colonies of some soil isolates showed positive results in the latex agglutination test, which was bought from Thailand. At the same time, some were found as weakly positive [18]. Next, they were cultured on Ashdown’s agar containing 10 g/L trypticase soy broth (Oxoid), 40 mL/L glycerol (Ultrapure), 5 mg/L 0.1% crystal violet (Sigma-Aldrich), 50 mg/L neutral red, 5 mg/L gentamicin (Gibco, Waltham, MA, USA) and 15 g/L agar. They incubated aerobically at 42°C for 4 days to assess if they could grow. They were kept in trypticase soy broth with 20% glycerol at -20°C.

Ethics review

The Siriraj Institutional Review Board approved the study (SIRB number: 546/2562 (EC1).

Confirmation for B. pseudomallei by 16S rRNA and recA sequencing assay

Bacterial DNA extraction was done using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. For 16S rRNA sequencing, we amplified a 1,488-bp-long fragment covering the whole 16S rRNA gene using the primers F229 (5-CGC AAG CGA AAG TAT CAA GA-3′) and R1908 (5-TTT ACA GCC GAT AAG CGT GAG-3′) according to a previously published article. We sequenced an 869-bp fragment of the recA gene using the previously published primers BUR1, 5-GAT CGA (A/G) AA GCA GTT CGG CAA-3′ and BUR2, 5-TTG TCC TTG CCC TG (A/G) C CG AT-3′. The PCR detection and mixture were described in a previous study [17]. Phylogenetic trees were constructed with each of 16S rRNA and recA sequences using MEGA software and analyzed the discriminatory power and type ability among the above gene sequencing assays.

Results

The 1,488-bp long nucleotide sequences of the whole length of 16S rRNA gene from 21 B. pseudomallei strains were investigated, aligned and compared. Eight different nucleotide positions (positions 157, 249, 651, 851, 968, 1232 and 1274) were found and no gaps were detected [18]. Among 23 16S rRNA genes of B. pseudomallei, 16S rRNA type 1 was probably identified in 15 clinical and 5 soil isolates (86.9%), while 1 clinical isolate (MMBP010) (4.3%) was probably 16S rRNA type 2 (Table 1) [18].

Table 1: Position of base differences in 16S rRNA type of studied B. pseudomallei.

Among the selected 15 soil isolates which were latex positive, 5 isolates were identified as B. pseudomallei (n=5) using 16S rRNA gene sequencing. In the case of the remaining 10 isolates, 16S rRNA gene sequencing can examine only one isolate (Letpandan_NMBt011) to be B. thailandensis (n=1) (Table 2). In contrast, the remaining 9 soil isolates showed contaminated peaks in the chromatogram of the DNA sequencing. For 14 soil isolates that were latex negative, 13 isolates were confirmed as B. thailandensis, while 1 isolate could not be identified. For 22 clinical Burkholderia isolates, B. pseudomallei (n=19) and B. cepacia (n=3) were identified by 16S rRNA gene sequencing in a previous study [17].

Table 2: Species identification by 16S rRNA and recA gene sequencing for soil isolates

A recA gene sequencing could discriminate all 45 soil and clinical isolates in contrast to 16S rRNA sequencing. Overall, a total of 29 soil isolates were investigated and there were B. pseudomallei (n=5), B. cepacia (n=2), B. cenocepacia (n=5), B. multivorans (n=4), and B. thailandensis (n=13). In contrast, recA gene sequencing presented higher discriminatory power than 16S rRNA gene sequencing. For 16 clinical isolates, recA gene sequencing identified B. pseudomallei (n=16) in the previous study [17].

Complete gene sequences of 16S rRNA (n=34) and recA (n=45) were determined in this study and recorded in GenBank with accession nos. CP041221.1 (B. pseudomallei), CP022217.1 (B. thailandensis), HM598390.1 to CP019678.1 (B. cenocepacia), LC487014.1 (B. cepacia) and HM598378.1 (B. multivorans) (Figures 1 and 2).

Figure 1: Phylogenetic analysis of clinical and environmental Burkholderia isolates in Myanmar by using 16S rRNA gene sequencing.

Figure 2: Phylogenetic analysis of clinical and environmental Burkholderia isolates in Myanmar by using recA gene sequencing.

Discussion

Tun Win and colleagues conducted a study in Myanmar to detect soil B. pseudomallei isolates using culture and latex agglutination tests [13]. The research was initiated during the hot season, following standard soil sampling procedures. However, during the wet season in this study, the true B. pseudomallei could rarely be identified, despite the isolates showing positive results in the latex agglutination after 16S rRNA and recA sequencing [19].

In contrast to this approach, this study utilized a latex agglutination test (4B11) purchased from the department of immunology and microbiology at tropical medicine. This test targeted the exopolysaccharide of B. pseudomallei and was used to screen Burkholderia species [20]. Further molecular identification of Burkholderia species, which showed positive agglutination tests, was conducted in the study. Given the resource constraints in Myanmar, the isolates were collected first and, next, performed molecular tests, identifying cross-reactive isolates capable of growing in Ashdown agar and exhibiting agglutination.

Interestingly, this study revealed that the identified strains expressed a B. pseudomallei-like capsular polysaccharide (BTCV), commonly found in Southeast Asian soil and remains nonpathogenic. This finding is consistent with the study conducted by Jirarat et al. The study highlighted the potential of utilizing molecular techniques to identify B. pseudomallei in resourcelimited settings such as Myanmar.

This study proposed a specific identification method for Burkholderia pseudomallei in Myanmar using 16S rRNA gene sequencing [17]. The agreement was found with the previous study by Gee et al., about using 16S rRNA gene sequencing for screening B. pseudomallei. The present study showed that the 16S rRNA gene sequences of Myanmar isolates were consistent with type I and II, revealing the same mutation point locations on a 1488 bp long gene fragment. The 16S rRNA type I incidence was higher in Myanmar isolates (90%) than type II, indicating a potential epidemiological tool for distributing B. pseudomallei.

However, further studies with more B. pseudomallei are needed to prove the representative samples. The long-range primer targeting the whole length of the 16S rRNA gene was used and no amplification of other soil Burkholderia species was observed. The variable region of the 16S rRNA gene was recommended as the target to improve identification accuracy.

Moreover, due to the inability of 16S rRNA gene sequencing to discriminate Burkholderia species from other soil species, recA gene sequencing was performed for identification [8]. This study was the first work in the identification of Burkholderia species in Myanmar. The findings agreed with a previous study in India, where recA gene sequencing showed higher resolution in identifying Burkholderia species than 16S rRNA gene sequencing by analyzing more clades of the phylogenetic tree in this study. Little is known about whether the recA gene of Burkholderia species in Myanmar is cross-reactive with other species, including Bordetella, Pandoraea, Ralstonia or Xanthomonas [8]. The proposed identification methods using 16S rRNA and recA gene sequencing could be used to identify B. pseudomallei and Burkholderia species in Myanmar, respectively. Further studies are needed to strengthen the findings and to investigate crossreactivity with other soil species.

Conclusion

This study showed that the latex agglutination test exhibited problems in accurate identification, screening or culturing among soil isolates. Therefore, additional confirmatory tests were required to isolate true B. pseudomallei in soil; in confirmation of B. pseudomallei, recA sequencing described a higher discriminatory power than 16S rRNA gene sequencing. This work provided the comparative assessment of sequencing among two genes, highlighting the correct choice for sequencing methods with cost-effectiveness in Myanmar.

Acknowledgments

We are grateful to all the laboratory practitioners in Myanmar who collected and stored the leftover samples. We express our gratitude to Mahidol neighboring countries grant's support for this publication. We are obligated to our colleagues working at the Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University.

Conflict of Interest

None to declare.

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