Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a trace for Burkholderia pseudomallei in Myanmar

Burkholderia pseudomallei; Enterobacterial repetitive intergenic consensus 5 (ERIC)-PCR, Multilocus sequence typing (MLST) 6 Authors: Nay Myo Aung,1,3* Khine Khine Su,3 Narisara Chantratita,2 Chanwit Tribuddharat1 7 Affiliations: 8 1Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, 9 Bangkok,10700, THAILAND 10 2 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol 11 University, 10400, THAILAND 12 3 Department of Microbiology, Defense Services Medical Academy, 11021, MYANMAR 13 *Corresponding author: Mailing address: Department of Microbiology, Defense Services 14 Medical Academy, 11021, Myanmar. Tel: +95 9262626644, E-mail: [email protected] 15 16 17 18 19 20 21 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 2

(196 words) 22 Melioidosis is a potentially fatal disease caused by Burkholderia pseudomallei , which is 23 endemic in Southeast Asia, including Myanmar. The typeability of enterobacterial repetitive 24 intergenic consensus (ERIC)-PCR assessed for 21 B. pseudomallei , they used the results of 25 sequence types (STs) of the multilocus sequence typing (MLST) method. Among 5 soil and 26 16 clinical B. pseudomallei isolates, the most significant bands were similar in position but 27 different in minor band formation. ST 90 of two soil strains (Tontae_NMBP001 and 28 Tontae_NMBP002) displayed the same ERIC banding pattern, while ST 56 of two clinical 29 isolates (MMBP005 and MMBP010) from different regions exhibited a single type. The same 30 ST found both clusters in the MLST method. The shared group STs showed four or three 31 satellite variants in the MLST scheme. One novel studied ST (ST 1729) and regarded it as an 32 out-group in the ERIC pattern. ERIC PCR demonstrated high discriminatory power, while 33 MLST provided more discrimination for genetic diversity. MLST requires extensive 34 sequencing and bioinformatics analysis, making it challenging to implement in resource-35 limited settings. More isolates are needed to validate these findings. Despite its limitations, 36 ERIC PCR represents a valuable and cost-effective alternative to MLST for molecular typing 37 of B. pseudomallei in resource-limited settings. 38 39 40 41 42 43 44 45 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 3 46

(2,488 words) 47 Melioidosis is an infectious disease caused by the gram-negative bacterium Burkholderia 48 pseudomallei, which is prevalent in the soil and water of Southeast Asia and Northern 49 Australia. The bacterium is an opportunistic pathogen that can cause a wide range of clinical 50 manifestations, from acute sepsis to chronic infections, with mortality rates as high as 40% 51 (1). Early diagnosis and prompt treatment with appropriate antibiotics are crucial for 52 successful outcomes; however, the accurate identification and typing of B. pseudomallei 53 remains challenging, particularly in resource-limited settings. 54 Molecular techniques have emerged as valuable tools for identifying and typing B. 55 pseudomallei isolates. Among these, enterobacterial repetitive intergenic consensus 56 polymerase chain reaction (ERIC PCR) and multilocus sequence typing (MLST) has widely 57 used for the molecular epidemiology and phylogenetic analysis of B. pseudomallei (2). ERIC 58 PCR is a PCR-based technique that amplifies the repetitive elements within the bacterial 59 genome, producing a DNA fingerprint that can use for strain typing and clustering analysis 60 (3). MLST, on the other hand, is a sequence-based method that targets specific genes in the 61 bacterial genome, enabling the identification of unique alleles and the determination of 62 genetic relatedness among isolates (4). 63 Despite the usefulness of MLST in identifying genetic variations and tracing the transmission 64 of B. pseudomallei, its implementation can be problematic in resource-limited settings due to 65 its high cost and technical requirements. In contrast, ERIC PCR is a simple and cost-effective 66 alternative method for the molecular typing of bacteria, including B. pseudomallei . This 67 technique amplifies the regions flanking the Enterobacterial Repetitive Intergenic Consensus 68 (ERIC) sequence, a repetitive DNA element in multiple copies in bacterial genomes. The 69 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 4 resulting banding patterns can be analyzed using gel electrophoresis, and clusters of strains 70 with similar patterns can be identified. 71 However, while ERIC PCR is a helpful tool for molecular epidemiology studies, it has some 72 limitations. For instance, it may not be as reliable as MLST in identifying genetically closely 73 related strains, as it depends on intergenic regions' variability rather than specific nucleotide 74 changes. Additionally, interpreting ERIC PCR results can be subjective, as the banding 75 patterns can be affected by experimental conditions and the interpretation of gel images (5). 76 Despite its limitations, ERIC PCR represents a valuable and cost-effective alternative to 77 MLST for molecular typing of B. pseudomallei in resource-limited settings. Its simplicity and 78 low cost could be available for surveillance and outbreak investigations, particularly in 79 endemic areas with limited advanced molecular methods. 80 In the context of Myanmar, where melioidosis is endemic, using these molecular techniques 81 to identify and type B. pseudomallei is essential for epidemiological investigations and 82 surveillance. However, the applicability of these methods in resource-limited settings needs 83 to evaluate. This study aims to provide an overview of the use of ERIC PCR and MLST for 84 identifying and typing B. pseudomallei in Myanmar and their potential as tools for the 85 surveillance and control of melioidosis. 86

87 Bacterial strain collection 88 Five soil and sixteen clinical isolates of Burkholderia pseudomallei were collected in a 89 previous study (6). Briefly, the published primers in the pudmlst website were used to 90 amplify the published housekeeping gene fragments ( ace, gltB, gmhD, lepA, lipA, narK, ndh) 91 (19, 158). ( https://pubmlst.org/bpseudomallei/). The PCR condition was evaluated in a 92 previous study, and continued amplicon sequencing was done using Sanger methods (First 93 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 5 Base company, Malaysia). Each isolate was analyzed by a string of seven integers (the allelic 94 profile), which correspond to the allele numbers at the seven loci, in the order ace-gltB-95 gmhD-lepA-lipA-narK-ndh. Next, each unique allelic profile was considered a clone and was 96 assigned a sequence type (ST), which also gave a convenient descriptor for the clone. An 97 MLST database containing the sequences of all alleles, the allelic profiles, and information 98 about the B. pseudomallei isolates, together with analysis tools, was recorded at Imperial 99 College (London, United Kingdom) and can be examined on the B. pseudomallei pages of the 100 MLST website (www.mlst.net). The resulting sequences at the seven loci were concatenated 101 in the order of loci used to determine the allelic profile. 102 For the genotyping of B. pseudomallei, we performed ERIC-PCR again, and a pair of forward 103 and reverse primers were used according to the reference article (3). The primers of 5'-ATG 104 TAA GCT CCT GGG GAT TCA C-3' (F) and 5'-AAG TAA GTG ACT GGG GTG AGC G-105 3' (R) were applied. The reaction was performed in a volume of in 20 μ l volumes containing 106 0.2 μ l of 1 U of DNA polymerase (Thermoscience), 1 μ l of DNA solution, 2 μ l of 1x standard 107 Taq reaction buffer (with MgCl2), 0.5 μ l of 0.25 mM each dATP, dCTP, dGTP, and dTTP, 108 and 0.5 μ l of 0.5 μ M each primer, adding 15.3 DNase free water. Finally, the thermocycler 109 was programmed. Simultaneously, negative ( Burkholderia species) were used to observe the 110

accurately. Gel bands of each isolate were examined under the installed software of 111 the gel documentation system. 112 Ethics review 113 The Siriraj Institutional Review Board approved the study (SIRB number: 546/2562 (EC1). 114

115 Among 21 isolates, ST 90 (n=6, 28.57%) was found as common ST from 3 clinical and soil 116 isolates, respectively (Table 1). The remaining isolates were resulted as previously published 117 and uploaded sequence types ST300 (n=1, 4.76%), ST 56 (n=2, 9.52%), ST 354 (n=2, 118 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 6 9.52%), ST 416 (n=1, 4.76%), which were isolated from clinical samples, whereas soil isolate 119 showed ST 42 (n=1, 4.76%). The rest 8 isolates were identified in novel STs, representing ST 120 1722, ST 1723, ST 1724, ST 1725, ST 1727, ST 1728, and ST 1729 from clinical samples 121 and ST 1726 from soil samples (Table 4.13). 122 As a resource-limited country, Myanmar, the rapid, cost-effective, and flexible genotyping 123

for B. pseudomallei isolates was developed, presenting the Enterobacterial Repetitive 124 Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) technique. Among 5 soil and 125 16 clinical B. pseudomallei isolates, it was seen that most of the major bands were quite 126 similar in position but different in minor band formation. Therefore, ST 90 of two soil strains 127 (Tontae_NMBP001 and Tontae_NMBP002) displayed the same ERIC banding pattern, while 128 ST 56 of two clinical isolates (MMBP005 and MMBP010) exhibited a single type. 129 Surprisingly, both of those two clusters were found to be the same ST in the MLST method 130 (Figure. 1). It is noteworthy to reveal that both clinical isolates with ST 56 were obtained 131 from patients residing in the same region of Yangon, which encompasses different cities such 132 as Hlegu and Khayan (Figure. 2). Overall, ST 90 were approximately analyzed as a same 133 clade, including one novel ST 1726. One novel ST (ST 1724) in this study was fo und in the 134 same cluster with old published ST 300 in global data, showing DLV difference in the MLST 135 scheme. It was observed that above mentioned 2 isolates exhibited 80% similarity in the 136 ERIC pattern. However, another novel STs in this study shared the same groups with 137 published STs (e.g., ST 1722 and ST 90, and ST 354 and ST 1725). The shared group STs 138 showed four or three satellite variants in the MLST scheme. One novel studied ST (ST 1729) 139 and was regarded as an out-group in the ERIC pattern. 140

141 MLST is a flexible and powerful epidemiological tool to study the distribution and evolution 142 of bacterial populations (7). ERIC PCR remains a rapid technique, easy to use, and cheap 143 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 7 with an acceptable outcome. However, it was still problematic in its reproducibility. 144 However, the quick assessment of B. pseudomallei was still essential due to its usefulness for 145 molecular epidemiology investigations in outreach areas and low-resource countries (8). In 146 this study, it was evaluated whether it was helpful to discriminate among STs of B. 147 pseudomallei. It was likely that it could identify shared groups among the same STs. Most 148 major band patterns of ERIC PCR exhibited approximately 80% similarity among historical 149 STs and novel STs of the present study. 150 This study found two isolates (MMBP005 and MMBP010) as a single genotype in the ERIC 151 PCR banding pattern. Surprisingly, those two isolates were isolated from different hospitals 152 with different regions but the same province and probably infected through traveling. There 153 was no assessment of STs from soil isolates, but an additional study should be conducted for 154 epidemiological research in the environmental association. Antonov et al. said that ribotyping 155 and pulsed-field gel electrophoresis are time-consuming and technically challenging for many 156 laboratories. ERIC PCR can be used for the rapid discrimination of B. mallei and B. 157 pseudomallei strains (9). In addition, detecting genetically diverse strains within a single 158 geographical area highlights the complex epidemiology of B. pseudomallei and the need for 159 continued surveillance and investigation of this pathogen in Myanmar. 160 For ST 90, two soil isolates were collected from the same region, but some clinical isolates 161 were distinct and showed the same clade. Interestingly, ST 90, which was observed to be a 162 part of a clade with one novel ST (ST 1726), exhibited approximately 80% similarity in the 163 ERIC pattern. This finding suggests that these strains may have a common ancestor and may 164 be related to each other. The presence of satellite variants in the MLST scheme for shared 165 group STs (e.g., ST 1722 and ST 90, and ST 354 and ST 1725) further supports the idea of 166 genetic diversity within these groups. 167 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 8 On the other hand, the novel ST 1729 was identified as an out-group in the ERIC pattern, 168 indicating that this strain may be genetically distinct from the different strains studied. 169 Further analysis is needed to determine the significance of this observation. A few isolates 170 that showed a single genotype in the present study were not representative of discrimination 171 of B. pseudomallei, and it pointed out for further research. 172

173 This study showed that ERIC PCR represents a valuable and cost-effective alternative to 174 MLST for molecular typing of B. pseudomallei in resource-limited settings. Its simplicity and 175 low cost make it an attractive option for surveillance and outbreak investigations, particularly 176 in endemic areas with limited advanced molecular methods. 177 Acknowledgments 178 We are grateful to all the laboratory practitioners in Myanmar who collected and stored the 179 leftover samples. We express our gratitude to Mahidol Neighboring Countries Grant's support 180 for this publication. We are obligated to our colleagues from the Department of 181 Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University. 182 Conflict of Interest 183 None to declare. 184 185 186 187 188 189 190 191 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint 9

192 1. Currie BJ, Ward L, Cheng AC. The Epidemiology and Clinical Spectrum of Melioidosis: 193 540 Cases from the 20 Year Darwin Prospective Study. PLoS Negl Trop Dis. 2010 Nov 194 30;4(11):e900. 195 2. Antonov VA, Tkachenko GA, Altukhova VV, Savchenko SS, Zinchenko OV, Viktorov 196 DV, et al. Molecular identification and typing of Burkholderia pseudomallei and 197 Burkholderia mallei: when is enough enough? Trans R Soc Trop Med Hyg. 2008 198 Dec;102 Suppl 1:S134-139. 199 3. Ranjbar R, Tabatabaee A, Behzadi P, Kheiri R. Enterobacterial Repetitive Intergenic 200 Consensus Polymerase Chain Reaction (ERIC-PCR) Genotyping of Escherichia coli 201 Strains Isolated from Different Animal Stool Specimens. Iran J Pathol. 2017;12(1):25–202 34. 203 4. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, Dijl JM van, Laurent F, et al. Overview of 204 molecular typing methods for outbreak detection and epidemiological surveillance. 205 Eurosurveillance. 2013 Jan 24;18(4):20380. 206 5. Bilung LM, Pui CF, Su’ut L, Apun K. Evaluation of BOX-PCR and ERIC-PCR as 207 Molecular Typing Tools for Pathogenic Leptospira. Disease Markers. 2018 Aug 208 1;2018:e1351634. 209 6. Aung NM, Su KK, Chantratita N, Tribuddharat C. Workflow for Identification of 210 Burkholderia pseudomallei Clinical Isolates in Myanmar. Jpn J Infect Dis [Internet]. 211 2022 Nov 1 [cited 2023 Mar 3]; Available from: 212 https://doi.org/10.7883/yoken.JJID.2022.508 213 7. Multilocus Sequence Typing and Evolutionary Relationships among the Causative 214 Agents of Melioidosis and Glanders, Burkholderia pseudomallei and Burkholderia mallei 215 | Journal of Clinical Microbiology [Internet]. [cited 2023 Mar 5]. Available from: 216 https://journals.asm.org/doi/full/10.1128/JCM.41.5.2068-2079.2003 217 8. ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-218 specific fragments as molecular markers to identify and track populations common to 219 healthy human guts - ScienceDirect [Internet]. [cited 2023 Mar 5]. Available from: 220 https://www.sciencedirect.com/science/article/abs/pii/S0167701204001691 221 9. Currie BJ, Dance DAB, Cheng AC. The global distribution of Burkholderia pseudomallei 222 and melioidosis: an update. Transactions of The Royal Society of Tropical Medicine and 223 Hygiene. 2008 Dec 1;102(Supplement_1):S1–4. 224 225 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint Figure 1 Assessment of ERIC patterns with related STs in Myanmar and highlight boxes showed the same patterns with the same STs The same clone was a color-coded group (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint Figure 2 Distribution of STs 56 in Yangon division (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint Table 1 Myanmar B. pseudomallei isolates analyzed by multilocus sequence typing Strain Source Year ST Allele profile Type of specimen ace gltB gmhD lepA lipA narK ndh MMBP001 Human blood 2018 300 1 1 3 1 1 4 1 MMBP002 Human blood 2018 1722 a 4 2 3 1 1 2 3 MMBP003 Human Urine 2018 1723 a 1 4 49 1 1 2 1 MMBP004 Human wound 2018 1728 a 1 12 6 1 10 4 1 MMBP005 Human blood 2018 56 3 1 4 1 1 4 1 MMBP006 Human tissue 2018 1724 a 1 1 3 1 8 2 1 MMBP007 Human blood 2018 1725 a 1 2 3 2 3 3 3 MMBP008 Human blood 2018 354 1 1 3 2 1 4 1 MMBP009 Human blood 2018 354 1 1 3 2 1 4 1 MMBP010 Human blood 2018 56 3 1 4 1 1 4 1 MMBP011 Human Urine 2018 1729 a 1 12 6 1 9 4 1 MMBP012 Human blood 2018 1727 a 1 12 13 2 1 1 3 MMBP013 Human pleural fluid 2018 416 1 12 6 1 1 4 1 MMBP014 Human blood 2018 90 1 12 6 1 1 4 1 MMBP015 Human pus 2018 90 1 12 6 1 1 4 1 MMBP016 Human blood 2018 90 1 1 6 2 1 42 1 Tontae_NMBP001 Soil Soil 2018 90 1 12 6 1 1 4 1 Tontae_NMBP002 Soil Soil 2018 90 1 12 6 1 1 4 1 Tontae_NMBP003 Soil Soil 2018 90 1 12 6 1 1 4 1 Pathein_NMBP004 Soil Soil 2018 42 1 12 6 2 1 2 1 Pathein_NMBP005 Soil Soil 2018 1726 a 1 10 6 2 1 2 1 /i1Showing novel ST (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 16, 2024. ; https://doi.org/10.1101/2024.02.15.580599doi: bioRxiv preprint