Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains.

BACKGROUND: Burkholderia pseudomallei is an environmental gram-negative bacterium that causes the disease melioidosis and is endemic in many countries of the Asia-Pacific region. In Australia, the mortality rate remains high at approximately 10%, despite curative antibiotic treatment being available. The bacterium is almost exclusively found in the endemic region, which spans the tropical Northern Territory and North Queensland, with clusters occasionally present in more temperate climates. Despite being endemic to North Queensland, these infections remain understudied compared to those of the Northern Territory. METHODOLOGY/PRINCIPAL
FINDINGS: This study aimed to assess the prevalence of central nervous system (CNS) disease associated variant bimABm, identify circulating antimicrobial resistance mutations and genetically distinct strains from Queensland, via comparative genomics. From 76 clinical isolates, we identified the bimABm variant in 20 (26.3%) isolates and in 9 (45%) of the isolates with documented CNS infection (n = 18). Explorative analysis suggests a significant association between isolates carrying the bimABm variant and CNS disease (OR 2.8, 95% CI 1.3-6.0, P = 0.009) compared with isolates carrying the wildtype bimABp. Furthermore, 50% of isolates were identified as novel multi-locus sequence types, while the bimABm variant was more commonly identified in isolates with novel sequence types, compared to those with previously described. Additionally, mutations associated with acquired antimicrobial resistance were only identified in 14.5% of all genomes.
CONCLUSIONS/SIGNIFICANCE: The findings of this research have provided clinically relevant genomic data of B. pseudomallei in Queensland and suggest that the bimABm variant may enable risk stratification for the development CNS complications and be a potential therapeutic target.

Introduction

Burkholderia pseudomallei is an environmental gram-negative pathogen present in Asia, South America, Africa and the Pacific. The resulting disease, melioidosis, is endemic across northern tropical Australia, with occasional clusters appearing in more temperate climates such as Western Australia, Southeast Queensland and New South Wales. Studies of melioidosis in Australia are disproportionally inclusive of the Top End of the Northern Territory and its surrounds. This geographic area is made up of approximately one quarter indigenous Australians, most of which live in small remote communities/towns [1]. Indigenous Australians in this area are significantly more affected and have a greater burden of disease [2,3], while melioidosis has also been reported as the leading cause of fatal community-acquired bacteremic pneumonia in the region’s largest hospital [1].

The disease melioidosis, caused by B. pseudomallei infection, can present with an extensive range of disease manifestations including, but not limited to, pneumonia, sepsis, and skin and soft tissue abscesses [4]. Successful treatment can be compromised by the presence of CNS disease (e.g. encephalomyelitis, abscess, meningitis, cranial nerve impairment) [5]. As these infections are often sub-clinical, exhibit varying times to clinical presentation, have diverse clinical presentations and affect multiple other organs; they can be challenging cases to manage [5,6].

CNS infections have been associated with the virulence factor bimA a variant of the wild-type, bimA [7-9]. These bimA genes encode as intracellular motility factor A, type V effector proteins that utilise host cellular actin. This motility results in the ability to evade the host immune system and invade the CNS via inter-cellular host cell migration [10]. The bimA variant is significantly truncated to that of bimA and is significantly more similar to bimA, of the highly infectious B. mallei, that is fatal in humans if left untreated [11]. Furthermore, the carboxy-terminals of the bimA genes of both species are also similar to the YadA auto-secreted adhesion protein of Yersinia enterocolitica, another highly pathogenic bacterial species [10,12,13]. Patients with infections possessing the bimA gene are suggested to be 14 times more likely to present with CNS disease, while the wild-type variant bimA is linked to pneumonia [7,14].

The bimA gene has been identified in both clinical and environmental isolates from the Northern Territory; yet isolates from North Queensland have not been as extensively characterised, despite both states being endemic regions [7,14] and tropical far north Queensland showing similar disproportionate patterns of infection and disease burden in indigenous Australian populations, to those reported in the Northern Terrritory [15,16]. Clinical presentations of CNS disease have been documented in a retrospective Queensland isolate collection [17]; however, the diversity of bimA and the association with central nervous system disease in Queensland remains unknown.

This study aimed to use comparative genomics to screen for the presence of the virulence factor bimA which has been previously linked to CNS disease in B. pseudomallei infections in patients of the Northern Territory. Furthermore, genomic diversity and antimicrobial resistance profiles are described.

Methods

Ethics statement

This study was performed under ethics approval from the Human Research and Ethics Committee of the Royal Brisbane and Women’s Hospital, as low or negligible-risk with waiver of patient consent (LNR/2020/QRBW/65573); site specific authority was obtained from the Townsville Hospital and Health Service with approval under the Queensland Public Health Act to access de-identified patient data.

Isolates

Clinical B. pseudomallei isolates have been collected prospectively over the last 22 years (1996–2018) from participating hospital pathology facilities (Cairns, Townsville and Central laboratories of Pathology Queensland). A total of 76 isolates were selected between postcodes 4895–4500 to include isolates from all of Queensland, from a collection of 400 clinical isolates. All isolates from patients with documented CNS involvement were included, which consisted of, six blood, four skin and soft tissue, four cerebral spinal fluid, two brain, and two sputum isolates(n = 18). The remaining 58 isolates were selected to include a variety of other common B. pseudomallei clinical presentations (Table 1). An additional 24 publicly available reference genomes were included in this study, of these, 13 were derived from the Northern Territory, eight from Queensland and one representative each from Papua New Guinea, China and Thailand (Table C in S1 Text).

Table 1

Metadata regarding the B. pseudomallei isolates included in this study.

B. pseudomallei isolates
Dates collected	1996–2018
Geographical range	4000–4895 Queensland, Australia
gender	31 F, 45 M
Age range	6–84 years
Central Nervous System Disease	18 (23.7%)
Mortality*	15 (19.7%)
Isolation sites:
Blood	42
Pus	8
Sputum	7
Cerebral spinal fluid	4
Tissue	4
Brain	2
Endotracheal aspirate	2
Bronchoalveolar Lavage	1
Gastrointestinal Tract	1
Liver	1
Lung aspirate	1
Lymph	1
Urine	1
Unknown	1
Total:	76

*only information for 23/76 isolates was available.

DNA extraction and WGS

The 76 isolates were recovered from -80°C storage and subcultured twice to ensure purity. DNA extraction was performed with the QIAGEN DNAeasy ultra-pure DNA extraction kit according to manufacturer’s instructions. Sequencing libraries were generated using the Nextera Flex DNA library preparation kit and sequenced on the MiniSeq System (Illumina Inc., San Diego, CA, USA) on a high output 300 cycle cartridge according to the manufacturer’s instructions. Five strains with unique BimA sequence and/or genomic diversity were prepped for long read sequencing using the MinION (Oxford Nanopore Technologies, Oxford, UK), where sequencing libraries were generated using the Rapid Barcoding Sequencing Kit (SQK-RBK004) and run on a flow cell R9.4.1 for 72 hrs. Data generated from this study is available under the NCBI accession PRJNA717363.

Genomic analysis

Illumina reads were trimmed with Trimmomatic v0.36 [18] and quality assessed with multiQC [19], genomes were assembled with SPAdes v3.14.0 [20] and annotated with Prokka v1.13 [21]. Long reads were filtered, quality checked, assembled and polished with Illumina reads where applicable using the MicroPIPE pipeline [22].

All read mapping was performed with BWA-MEM [23]. Sequence types (STs) were determined with multi-locus sequence typing (MLST) [24] (https://github.com/tseemann/mlst) and reads mapped to alleles retrieved from pubMLST, where SNPs were suspected [24]. Genotypic antimicrobial resistance was determined with ArDaP [25]. Whole genome alignment (4,950,632bp) and phylogenomic analysis of the 24 reference genomes and 76 genomes derived from this study (n = 100) were achieved using parSNP [26] JModelTest[27] (TVM+F+I+G4) and IQtree [28] (1000 bootstrap (BS) replicates).

Statistical analysis

Using Stata and the csi command for unstratified cumulative incidence data [29], and isolates with bimA or CNS presentation (n = 29), Odds Ratio and Chi-Square were calculated, with a P value <0.05 considered significant.

Results

In total, we identified the bimA virulence factor in 20/76 Queensland isolates (26.3%), geographically limited to the northern half of Queensland (Fig 1). Of the 18 isolates with known CNS disease, nine were found to carry the bimA virulence factor and nine carried the wild type bimA. A total of 20 isolates were confirmed to carry the bimA virulence factor, of which eleven had no clinical record of CNS disease. Risk analysis supported bimA being associated with CNS infection (Odds Ratio 2.8, 95% CI 1.3–6.1, P = 0.009).

Fig 1

Distribution of B. pseudomallei isolates with the bimA virulence factor in north Queensland (Generated using the rnaturalearth package in R: https://cran.r-project.org/web/packages/rnaturalearth/README.html).

The variation observed in the 20 bimA protein sequences was significant amongst isolates, with 18 proteins possessing unique sequences, while none of the sequences produced in this study were identical to that of the reference strain MSHR668. The variation was observed almost only in the proline-rich region of the protein (90-160aa) (Fig 2). However, five isolates carried an amino acid variant ΔN213S (Fig 2A, TSV1, TSV152, TSV141, TSV164, CAM60), of which four isolates had documented CNS disease (one individual died before diagnosis, CAM60), and four cases resulted in death (one individual made an unexpected recovery while in intensive care, TSV 152). These isolates were collected from as early as 1996, with the latest in 2012 and were significantly geographically dispersed (350–850 km apart). Additionally, four of these same isolates (TSV1, TSV152, TSV164, CAM60) and isolate TSV294 also possessed an upstream mutation ΔR11H (Fig 2B). Additionally, all isolates carrying bimA also possess a truncated bimC gene which plays a role in intracellular spread and lies upstream of bimA (S1 Fig).

Fig 2

Alignment of the BimA proline-rich region identified in Queensland B. pseudomallei isolates.

Extensive variation of the proline rich region coloured green, is observed between isolates included in this study as well as compared to the Queensland BimA reference genome MSHR668 located at the top of the alignment. Fig 2A. Alignment depicting the four isolates carrying an amino acid variant ΔR11H upstream of the proline rich region of the BimA gene. Fig 2B. Alignment depicting the five isolates carrying an amino acid variant ΔN213S downstream of the proline rich region of the BimA gene.

From the 76 isolates we were able to identify 27 known multi-locus sequence types (STs) from 38 isolates (50%) (Table A in S1 Text). From these, at least three sequence types (109, 151, 1667) have also been identified in the Northern Territory. Of the 38 novel sequence types, 26 isolates were comprised of previously described alleles in a novel combination, and 12 were due to a SNP present in one of the seven alleles (Fig 3 and Table B in S1 Text). Novel allele variants were identified across six of the alleles, with the allele ace the only one without any variants (n = 12). In each novel case, sequence types were confirmed via read mapping to all seven alleles. In total 34 of the 38 isolates carried unique novel STs, as two of the STs occurred twice and one ST occurred three times in the sample set. Among the 20 isolates carrying the bimA variant, 13 (65%) carried novel STs, over previously described STs (n = 7, 35%; Fig 3).

Fig 3

Maximum likelihood phylogenomic tree of Australian B. pseudomallei isolates, rooted to reference MSHR668.

Bootstrap support ≥80 is shown at nodes in red and reference genomes are labelled with strain name stated in NCBI. Tree was built using IQtree 100 genomes and a 4,950,632 bp alignment generated from parSNP.

Phylogenomic analysis revealed the majority of Queensland isolates with novel STs branched significantly closer to the Queensland bimA reference genome MSHR668, than to the Northern Territory and Asian genomes. This was also true for the bimA variant. Two separate lineages can be identified in the phylogeny (point of divergence is approximately in the middle of the phylogeny), with the three clades at the bottom half of the phylogeny carrying 33 of the 38 novel STs identified (86.8%) and 18 of the 20 bimA variants present in the sample set (90%). MLST diversity did not appear to be influenced by or correlated with the date the isolate was collected (Fig 3). No Queensland isolate was identical to that of a Northern Territory isolate, with limited clustering with Northern Territory or other Australian isolates. Additionally, five of the Queensland isolates included in this study clustered within the Australian/Asian clade. In this clade the reference genome K96243 from Thailand is the most basal, with BPC006 from China branching within the Australian isolates CAM2, 112, 189, TSV38 and 95. All of the isolates within this clade were of known ST and did not carry the bimA variant.

Approximately one third of the genomes did not contain any AMR related mutations at all (35.5%). The pre-cursor mutation to imipenem resistance (penA ΔT153A) was identified in 57.9% of all isolates. Only mutations associated with ceftazidime and meropenem resistance were predicted within the sample set. Ceftazidime resistance (loss of the PBP3 homolog BPSS1219) was only predicted from one isolate (1.3%), while meropenem resistance was predicted to be caused by the loss of function of the amrR efflux pump (BPSL1805) in 11 of the 76 isolates (14.5%). Meropenem resistance was encoded in 15% of bimA isolates and 16% of bimA isolates, respectively (Table 2).

Table 2

Comparison of clinical presentation, sequence type and antimicrobial resistance within bimA or bimA-carrying B. pseudomallei isolates.

Variable	bimABm Isolates (n = 20, (%))	bimABp Isolates (n = 56, (%))
CNS presentation	45	16
Novel ST	40	53.6
Known ST	60	46.4
amrR mutation	15	16
Pre-curser mutation	45	64.3
No AMR mutation	35	35.7
Mortality*	40	12.5

*information was only available for 23/76 isolates

Discussion

Variation in the bimA gene was present and extensive. Of the 76 isolates, 20 carried the virulence factor bimA and only two of the sequences produced from this study were identical (TSV39, TSV25). No sequence generated from this study was identical to any of the previously described reference sequences, suggesting depth of variation in the bimA gene is extensive and yet to be seen (Fig 2). Since the 2000’s sequence variation of BimA in B. pseudomallei has not been discussed in great detail, despite being studied recently [8]. For example, five isolates possessed a ΔN213S substitution, positioned between the proline rich region and the YadA-like head domain, a highly conserved region [9]. Interestingly, the isolates with this mutation had a mortality rate of 80% and documented CNS disease at a rate of 80% (one patient died before CNS disease could be diagnosed). Unfortunately, similar to previous studies the effect these variants have upon the function of the protein and resulting virulence remain unclear [9]. Reference sequences from MSHR33, 491 and 172 all possess the mutation as well, suggesting this is not a recent event [9]. However, such high mortality rates and disease severity suggests that isolates with this type of mutation may be more virulent than those without.

Previous studies have suggested that bimA carrying isolates are 14 times more likely to develop CNS disease than the wild-type bimA isolates [7]. Explorative analysis of this data suggested the bimA variant and the development of CNS disease to have an Odds Ratio of 2.8 (95% CI 1.3–6.1 P = 0.009), compared to the wild type. However, as this dataset was selected for particular factors (CNS disease and postcode) these numbers should be viewed with caution. Perhaps what is more representative of the Queensland population is the 11 bimA variants identified from the 58 non-CNS disease isolates, suggesting 19% of B. pseudomallei infections will carry the virulence factor. Indeed, larger sample sizes collected at random will be needed to confirm these numbers and this should be considered for future studies. Sarovich et al., also assessed the likelihood of clinical co-variants that may be associated with CNS disease, but did not find other associations [7]. Due to the lack of complete clinically relevant data surrounding the isolates in this study, we were unable to calculate if this was applicable to our results.

In this study, CNS disease isolates were evenly split between bimA and bimA genes and more isolates presented without CNS disease and BimA (n = 11), than with both of these variables (n = 9). Therefore, it is likely this gene is not the only factor driving the development of CNS disease. This was also evident in the Northern Territory, with 85.5% of isolates carrying the variant, not diagnosed with CNS disease. Additionally, 1% presented with CNS disease, but no variant [7]. In both studies this is a significant proportion of bimA isolates that have not developed CNS disease. An environmental study [14] of both bimA and another virulence factor lipopolysaccharide (LPS) suggested the bimA virulence factor was more likely to occur with LPS genotype B, the more prevalent genotype in Australia. Genotype A, is more prevalent in Thailand and Southeast Asia, where CNS disease is less frequently reported [7,14]. Furthermore, the gene upstream of bimA, bimC was also assessed in this study. As both bimA and bimC genes have been shown to play a role in the intracellular spread of B. pseudomallei [30]. As all bimA isolates in this study possessed a truncated bimC gene, the affect this truncation has upon virulence and the role of bimC in intracellular spread remains unknown. However, it suggests the variant is significant to virulence in these strains and should be further investigated. There is no way to show a definitive correlation between these two virulence factors and bimA in Queensland without including both clinical and environmental isolates in future studies. The authors suggest a pan-genomic analysis coupled with extensive clinical metadata from as many isolates as possible representing both states evenly, to be the best approach at identifying all genes associated with CNS disease. It is possible that there may be some combination of genes derived only from the unique genetic diversity of Australian isolates responsible for the increased incidences of CNS disease observed here in Australia compared to those overseas.

Although Queensland has been included in MLST diversity analyses before, novel STs have not been described, which is significant given 50% of isolates in this study were novel STs (Fig 3) [31,32]. A large number of isolates (34%) were comprised of a previously undescribed combination of alleles to generate a novel ST (Table B in S1 Text). It also appears that isolates with the bimA virulence factor are more likely to be novel STs. This implies that Queensland isolates are exchanging genetic material [32]. The exchange of genetic material was also evidenced by the lack of clonality seen in both previously described and novel STs in Queensland. ST 70 was the most commonly described (6.5%) and was identified in both Brisbane and Cairns isolates suggesting one ST does not dominate certain spatial areas in this study, despite this being reported for the Northern Territory [14,31]. Furthermore, the same studies reported that Queensland and the Northern Territory do not share any common STs. However, in this study we identified at least three STs shared between the states (STs 109, 151, 1667), with the potential of more, as the exact location was not recorded for all STs in pubMLST. The identification of shared STs here, may be due to the previous under-representation, or exclusion of Queensland in B. pseudomallei isolates in previous studies [31-34].

Genomic analyses also identified the loss of function of the amrR regulator (amrAB-OprA efflux pump), associated with meropenem resistance in 14.5% of isolates, however this has not been validated phenotypically [25]. A pre-curser mutation in penA ΔT153A, was common amongst the isolates and in combination with other missense and promoter mutations can cause imipenem and amoxicillin-clavulanic acid resistance [25]. This is not overly worrisome as imipenem is not used to treat B. pseudomallei in Australia, meropenem is the preferred carbapenem, however amoxicillin-clavulanic acid is sometimes used in eradication therapy [6]. Phenotypically, antimicrobial resistance circulating in the Northern Territory comprises of approximately 3% for doxycycline and 0.9% for trimethoprim-sulfamethoxazole [35], while isolates remain susceptible to meropenem and ceftazidime [4,35,36]. This data suggests AMR may be rarely encountered in Queensland isolates and the current selection of antimicrobials for use against B. pseudomallei infections will be effective.

Conclusion

This study has revealed a significant amount of genetic diversity in Queensland B. pseudomallei isolates, such as novel MLST sequence types and unique bimA gene sequences. The virulence factor bimA is linked to CNS disease, yet it is suspected there are multiple drivers for this type of infection. Further exploration into the virulence factors responsible for CNS disease should focus on maximum sample size, pan-genomics, detailed clinical metadata and include environmental samples. Identification of CNS disease drivers may act as a screening test to warn clinicians and prompt additional investigations such as a lumbar puncture or MRI or ultimately provide a novel therapeutic target.

Supplementary tables.

Table A in S1 Text. Known sequence multi-locus types identified in Queensland B. pseudomallei isolates. Table B in S1 Text. Novel Queensland B. pseudomallei multi-locus sequence types, ~ flags the closest known allele. Table C in S1 Text. Details of the Burkholderia pseudomallei reference data used in this study.

(DOCX)

Click here for additional data file.

Read mapping of bimC demonstrating truncation in isolate TSV2 compared to bimC of reference genome BPSS1491.

(PNG)

Click here for additional data file.

15 Nov 2021

Dear Dr. Harris,

Thank you very much for submitting your manuscript "Clinical Burkholderia pseudomallei isolates from Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Adly M.M. Abd-Alla, Prof asso.

Associate Editor

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Abdallah Samy

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: METHODS: How did the investigators select the control isolates (e.g., Queensland NON-CNS isolates)? Couldn't some apparent differences be introduced between the two groups simply by differences in era of enrollment, location within the geographically large state of Queensland, or even characteristics of the patient (immigrant status, race, gender, age, immunocompromise/diabetic state)?

Reviewer #2: Materials and Methods section for this study is straight forward. No issues.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results are clear and clearly presented.

Reviewer #2: #1) Lines 88-90 and Table 1. Which of the 18 isolates make up the CNS strains? 4 of the strains from CSF and 2 from the brain, but it was not clear where the other 12 come from as listed in Table 1. Also, on line 89, it lists 18 CSF strains and the remaining 57 but that adds up to 75. Line 86 refers to a total of 76 isolates.

#2) Figure 2. This reviewer would expand the legend description. It is not clear what I should be focused upon. Do the various colors represent something notable?

#3) Line 132. Points out the important amino acid variant N213S for the 5 strains. Should this be included/shown in Fig 2?

#4) Lines 136-139. These results are discussed as being important but not shown.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: 259-262 significant amount of genetic diversity in Queensland B.

265 unclear if absence of bimABm could identify low risk of CNS involvement given only half of the patients with CNS infection had the variant

Reviewer #2: No comments for conclusions.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: #1. Line 37. Should multi-locus sequence types be abbreviated as MLST versus ST? In other places (line 259), the abbreviation is shown as MLST.

#2. Table 2. Title refers to bimAps. Should this be bimABp?

#3. Line 176. Should this be worded: “Twenty isolated carried the “altered” virulence factor”?

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This study aimed to identify prevalence of bimABm, which is associated with meningitis/CNS infection, with comparative genomics ,

has been previously linked to neurological disease in B. pseudomallei infections in patients of the Northern Territory

From the 76 isolates analyzed,the bimABm variant was identified in 20 (26.3%) isolates and 9 (45%) of the isolates with documented central nervous system infection (n=18).

A significant association was noted between isolates carrying the bimABm variant and central nervous system disease (OR 2.8, 95% CI 1.3-6.0, P=0.009) compared with isolates carrying the wildtype bimABp.

This association, however, has been described for over a decade (cit. 7 Stevens Mol Microbiol 2005)

METHODS: How did the investigators select the control isolates (e.g., Queensland NON-CNS isolates)? Couldn't some apparent differences be introduced between the two groups simply by differences in era of enrollment, location within the geographically large state of Queensland, or even characteristics of the patient (immigrant status, race, gender, age, immunocompromise/diabetic state)?

120-126 Half of CNS isolates had bimABm; implying that it is not fully causative.

More interesting is what homologous genes were present in the 9 isolates from CNS diseas who did NOT have the bimABm variant.

Further, why did the authors not perform in vitro phenotypic testing to establish phenotypic variation in host actin-mediated mobility between the identified variants?

Alternately they could perform in vivo RNA seq on the bacteria to evaluate potential upstream regulatory differences among the bim ABm variants NOT associated with meningitis; and also among the 9 meningitis-derived isolates that did not manifest the BimABm variant.

151-154 Unsurprising result given proximity of the regions. More interesting are the Thailand derived isolates. Could these have been described amongst recent immigrants

167-172 Why not phenotypic confirmation of the AMR findings?

259-262 significant amount of genetic diversity in Queensland B.

265 unclear if absence of bimABm could identify low risk of CNS involvement given only half of the patients with CNS infection had the variant

Reviewer #2: The results are interesting but the manuscript could be tightened in a few areas to better relay the story. Details below.

#1) This reviewer feels more details, distinction, and appropriate references should be provided for bimAbm and bimABp.

a. The description is limited to just one sentence lines 63-67. This section lacks a description of what the difference are in general between these two versions of bimA.

b. Also, it may be a semantics issue, but is it appropriate to call this a mutant? Perhaps more appropriate would be variant or referring to those Bp strains with bimABm as a subset of a population.

c. The sentence starting at line 63 should have an appropriate reference for it.

#2) As highlighted above, many of the relevant mutations discussed in the results were not shown in the figures or even listed as "data not shown".

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Reviewer #2: No

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3 Jan 2022

Submitted filename: Response to reviewers commentsv2.docx

Click here for additional data file.

7 Mar 2022

Dear Dr. Harris,

Thank you very much for submitting your manuscript "Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Adly M.M. Abd-Alla, Prof asso.

Associate Editor

PLOS Neglected Tropical Diseases

Abdallah Samy

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: (No Response)

Reviewer #3: 1. The overall study design is appropriate and adequately address the hypothesis.

2. My only concern is if the authors have conducted a simple antibiotic susceptibility test on the isolates, particularly those 76 clinical isolates. This simple experiment can perhaps give a clearer correlation to AMR related mutations analysis conducted by the authors.

3. Line 98 - Papua New Guinea

Reviewer #4: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes

-Is the study design appropriate to address the stated objectives? Yes

-Is the population clearly described and appropriate for the hypothesis being tested? Yes even if some missing data about the strains could have been vey important

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? Yes

-Were correct statistical analysis used to support conclusions? Yes

-Are there concerns about ethical or regulatory requirements being met? Not applicable

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: (No Response)

Reviewer #3: 1. The results are clear and well presented.

2. Can the authors prove the correlation between the presence of AMR related mutations with the isolates phenotype (antibiotic susceptibility)?

Reviewer #4: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

-Are the figures (Tables, Images) of sufficient quality for clarity? Yes

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: (No Response)

Reviewer #3: The conclusions is well presented.

Reviewer #4: -Are the conclusions supported by the data presented? Yes

-Are the limitations of analysis clearly described? Could be improved

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? Yes

-Is public health relevance addressed? Yes

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: Several minor editorial suggestions for the authors.

Lines 35 & 59. Add abbreviate for CNS as needed.

Line 51. ....Territory and surrounds. Is something wrong/ missing with sentence?

Line 51. This geographic.... Should "area" be added?

Line 57. Something missing from the start of this sentence? "Infections cause the disease meliodosis....

Line 68. bimAMa- is this supposed to be bimABm?

Line 74. I would recommend saying which gene we are speaking of bimA, bimABp, or bimABm?

Line 151. What is "ace"?

line 186 &211. This is gene, correct? Should be "b".

Reviewer #3: Minor revision

Reviewer #4: Line 24: with antibiotic treatment?

Line 74: precise the version of the gene

Line 223-224: could be interesting to study this with mutants and wildtype infections

Line 238: are the alleles related to virulence genes?

Table 2: problem in the percentage column

Figure 2: legend title ‘Alignment of…’

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: Previous concerns addressed. Just minor editorial items for the authors' consideration.

Reviewer #3: The paper is suitable for publication.

Reviewer #4: The study described in this paper is interesting as it presents the situation of melioidosis in a part of Australia and asociated variants/mutations and also virulence and antimicrobial resistance. The study is well supported by the data included even if some missing data about the strains could have been of great importance to trace back the origin of the strain (but it is sometimes difficult, we all know that).

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

20 Apr 2022

Submitted filename: Response to reviewers comments v4.docx

Click here for additional data file.

24 May 2022

Dear Dr. Harris,

We are pleased to inform you that your manuscript 'Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

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Adly M.M. Abd-Alla, Prof asso.

Associate Editor

PLOS Neglected Tropical Diseases

Abdallah Samy

Deputy Editor

PLOS Neglected Tropical Diseases

8 Jun 2022

Dear Dr. Harris,

We are delighted to inform you that your manuscript, "Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases