Genome-wide association study of antibiotic resistance in clinical Burkholderia pseudomallei strains from Hainan province, China

doi:10.21203/rs.3.rs-3140673/v1

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Genome-wide association study of antibiotic resistance in clinical Burkholderia pseudomallei strains from Hainan province, China

https://doi.org/10.21203/rs.3.rs-3140673/v1

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Background

Burkholderia pseudomallei is a gram-negative soil-borne Bacillus pathogen that causes melioidosis and poses a significant threat to human health. This study aimed to investigate pan-genetic resistance genes using the genome sequences of 556 clinical strains. The predominant resistant phenotypes observed among these strains were resistance to DOX and AMC.

Results

We identified OprM and MexB as genes significantly associated with DOX-resistant phenotypes. Moreover, gene ontology (GO) enrichment analysis was performed to explore the functional implications of the genes found in the DOX and AMC-resistant strains, indicating their involvement in the negative regulation of cellular and carboxylic acid biosynthetic processes. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed distinct pathways enriched in DOX-resistant strains, namely oxidative phosphorylation and glutathione metabolism, whereas AMC-resistant strains exhibited enrichment in lysine biosynthesis; valine, leucine, and isoleucine degradation; and, amino acid and nucleotide sugar metabolism pathways.

Conclusions

This study helps to understand the evolution of drug resistance in this bacterium and potential targets for predicting drug resistance, and provides a foundation for clinical diagnosis within hospital microbiology and medical facilities.

Burkholderia pseudomallei

antibiotic resistance

genomics

whole-genome sequencing

mechanism

Burkholderia pseudomallei is a gram-negative, soil-borne bacillus that is mainly distributed in Southeast Asia and Northern Australia. It is one of the most frequently found microorganisms in clinical environments and causes melioidosis worldwide [1–3]. People are primarily infected by contaminated soil and water [4]. A report on 44 melioidosis cases was identified from a hospital in Zhanjiang, of whom 25 (56.8%) died [5]. The high mortality rate due to the antibiotic resistance and wide distribution of B. pseudomallei has made it a life-threatening pathogen that is difficult to treat [6, 7].

Antibiotics therapy is the most common solution for B. pseudomallei infections in clinical settings. Nevertheless, B. pseudomallei exhibits intrinsic resistance to numerous antibiotic families, such as aminoglycosides, certain β-lactams, and macrolides, thereby posing a significant challenge to human health [8–10]. Therefore, it is important to explore the pathogenic characteristics of antibiotic-resistance-genes in the whole genome of B. pneudomallei to strengthen surveillance and control the spread of this pathogen.

Genome-wide association studies (GWAS) have recently been developed as the most effective method for analyzing the relationship between bacterial phenotypes and genetic variants [11, 12]. However, the precise mechanisms by which genetic variations influence the development of antibiotic resistance in B. pseudomallei have not been extensively investigated. Recently, a novel approach was employed to discern the presence of single nucleotide polymorphisms (SNPs) within the DNA, potentially associated with β-lactam resistance in Streptococcus pneumoniae [13, 14]. Furthermore, whole-genome sequencing (WGS) was employed to comprehensively analyze a dataset comprising 1,680 S. pneumoniae strains obtained from four distinct populations. This investigation successfully identified potential genetic hotspots exhibiting significant variation correlated with phenotypic resistance to indispensable classes of antibiotics [15].

With the accumulation of specific genes or SNPs prior to full antibiotic resistance in suitable clones, WGS and GWAS could be effective ways to assess the rate of accumulation of potential resistant variants and provide an advanced prediction signal for increased antibiotic resistance. In this study, we used genome sequencing and GWAS to explore SNPs and genes associated with resistance to two different antibiotics, jointly referred to as antibiotic resistance. The accumulative effect of B. pseudomallei along with these resistance-conferring variants among the whole genome is called “distance to resistance”. Accordingly, a genotypically diverse group of 556 clinical B. pseudomallei strains from Hainan province was analyzed using GWAS to elucidate the underlying mechanism of antibiotic resistance in the whole genome. These findings provide new insights for therapeutic options against B. pseudomallei infections in hospitals.

Pan-genome of clinical B. pseudomallei strains

Annotation and pan-genome analysis of the B. pseudomallei genome identified 23,060 genes, of which 2,621 were core genes and 648 were soft core genes (Fig. 1A). With an increase in the number of genomes, the total number of genes in the pan-genome increased, but the number of core genes remained unchanged (Fig. 1B). The increasing trend of the total genes indicated that the pan-genome of B. pseudomallei was open (Fig. 1B). Antibiotic resistance genes were further predicted, and 30 relevant resistance genes were finally identified in the comprehensive antibiotic resistance database (CARD), of which 14 genes were associated with tetracycline resistance, including MexK, MexB, OprM, MexI, MuxB, MuxC, MexA, and OprA (Table 1).

Antibiotic resistance phenotypes of 1,680 B. pseudomallei strains

Drug susceptibility assays were performed for 11 antibiotics, including ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), meropenem (MEM), doxycycline (DOX), trimethoprim-sulfamethoxazole (SXT), tazobactam (TZP), imipenem (IPM), chloramphenicol (CHL), cefoperazone-sulbactam (SCF), tigecycline (TGC), and ciprofloxacin (CIP). The results indicated that some isolates showed resistance to AMC, DOX, SXT, and MEM, but all of them were sensitive to other drugs (Table 2). Among them, resistant isolates were identified for AMC (four resistant and 19 intermediately sensitive), DOX (five resistant and 33 intermediately sensitive), and SXT (five resistant) according to the Clinical and Laboratory Standards Institute, 2015 (CLSI) (Table 2). The distribution of AMC-, DOX-, and SXT-resistant strains was scattered throughout the phylogenetic tree (Fig. 2).

Accessory genes associated with DOX and AMC resistance

Accessory genes significantly associated with resistant phenotypes were detected by pan-GWAS, in which 1,658 genes (odds ratio (OR) > 1) were present and 1,007 genes (OR < 1) were absent in AMC-resistant strains (Fig. 3A). Meanwhile, 665 genes (odds ratio > 1) were present and 566 genes (odds ratio < 1) were absent in the DOX-resistant strain (Fig. 3B). The genes significantly associated with the AMC-resistant phenotype included the known resistant genes, group_2287 (antibiotic efflux pump outer membrane protein, ArpC) and group_4353 (multidrug resistance protein, MdtC) (Table S2). The genes significantly associated with the DOX-resistant phenotype included group_693 (OprM), group_2285 (OprM), and group_22059 (MexB), which were predicted by Abbricate and carried in a significantly higher proportion in resistant strains than in sensitive strains (Table S3).

Functional analyses of antibiotic-resistant genes

Genes significantly associated with the DOX- and AMC-resistant phenotypes were further subjected to Gene Ontology (GO) (Fig. 4) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) (Fig. 5) enrichment analyses. GO enrichment analysis showed that the genes present in the DOX- and AMC-resistant strains were involved in the negative regulation of cellular processes (Fig. 4A) and carboxylic acid biosynthetic processes (Fig. 4C), respectively. The genes absent in the DOX-resistant strains were mainly enriched in cytoplasmic ribonucleoprotein granules, pole plasma, and germ plasma (Fig. 4B). Genes absent in AMC-resistant strains were associated with cells and cell parts (Fig. 4D). KEGG enrichment analysis showed that genes present in the DOX-resistant strains were enriched in oxidative phosphorylation, Streptomyces biosynthesis, and glutathione metabolism pathways (Fig. 5A). The genes absent in the DOX-resistant strains were mainly enriched in porphyrin metabolism, thiamine metabolism, pentose and glucuronate inter conversions, peptidoglycan biosynthesis, and fructose and mannose metabolism pathways (Fig. 5B). The genes present in AMC-resistant strains were enriched in lysine biosynthesis; thyroid hormone synthesis; valine, leucine, and isoleucine degradation; and amino acid and nucleotide sugar metabolism pathways (Fig. 5C). The genes absent in AMC-resistant strains were mainly enriched in porphyrin metabolism, biosynthesis of siderophore group non-ribosomal peptides, photosynthesis, peptidoglycan biosynthesis, and biosynthesis of ansamycin pathways (Fig. 5D).

We further correlated the presence of antibiotic-resistant genes with the genetic distance between the bacterial strains. The results showed that DOX resistant genes, including group_693, group_2285 (OprM), and group_22059 (MexB) (Fig. 6A), and AMC resistant genes such as group_2287 (ArpC) and group_4353 (MdtC) (Fig. 6B) were relatively scattered in the phylogenetic tree of the strains.

This study included 556 B. pseudomallei isolates from the Hainan province in China. Of these, 23,060 genes and 2,621 core genes were identified using pan-genome analysis. Among these strains, 17 antibiotic resistant genes and 8 genes (MexK, MexB, OprM, MexI, MuxB, MuxC, MexA, and OprA) were associated with tetracycline resistance as predicted by the CARD. It has been shown that efflux is the dominant mechanism of antibiotic resistance in B. pseudomallei [16, 17]. Meanwhile, Mex-family and Opr-family proteins constitute a functional efflux pump and play an important role in the mechanism of antibiotic resistance [18, 19]. Among these, four efflux pumps most significantly contribute to drug resistance: MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM [20]. A previous study showed that overexpression of efflux pumps MexB and MexY significantly increased antibiotic resistance in clinical Pseudomonas aeruginosa[21].

The drug sensitivity test results showed that the majority of the resistant strains were resistant to AMC (4 resistant and 19 intermediately sensitive), DOX (5 resistant and 33 intermediately sensitive), and SXT (5 resistant). A previous study showed that all B. pseudomallei isolates from Bangladesh were consistently sensitive (100%) to CAZ, IPM, piperacillin–tazobactam, AMC, and tetracycline using both disk diffusion and minimal inhibitory concentration (MIC) methods [22]. Therefore, it is necessary to explore effective approaches for controlling drug resistance in B. pseudomallei.

Based on pan-GWAS analysis of the whole genome of B. pseudomallei, the genes significantly associated with the AMC resistant phenotype were the known resistant genes group_2287 (ArpC) and group_4353 (MdtC). Outer membrane efflux pump proteins play a significant role in various resistance mechanisms acquired by Acinetobacter baumannii [23, 24]. The genes significantly associated with the DOX-resistant phenotype were group_693 (OprM), group_2285 (OprM), and group_22059 (MexB). A previous study has shown that MexAB-OprM pump contributes to a channel-forming system which allows the extrusion of drugs from the periplasm directly into the extracellular environment, resulting in clinical antibiotic resistance [25–27]. Our pGWAS results were consistent with those of previous studies, and functional research on these drug resistant genes should be performed. Moreover, the results showed that the distribution of DOX- and AMC-resistant genes was relatively scattered in the phylogenetic tree of the strains. This indicates that horizontal gene transfer may play a vital role in the evolution of drug resistance in B. pseudomallei. Previous studies showed that incoming DNA or RNA by horizontal gene transfer could replace existing genes or introduce new genes into the genome [28, 29]. Thus, the acquisition of bacterial drug resistance by horizontal gene transfer may be influenced more by the genetic background.

According to the GO enrichment analysis, the DOX- and AMC-resistant strains were involved in cellular and carboxylic acid biosynthetic processes. A previous study showed that the predicted biosynthesis of bogorol starts with two non-ribosomal peptide modules that introduce 2-hydroxy-3-methylpentanoic acid (Hmp), a carboxylic acid that is highly associated with bacterial drug resistance [30]. The KEGG enrichment analysis demonstrated that DOX-resistant strains were involved in oxidative phosphorylation, streptomyces biosynthesis and glutathione metabolism pathways. Previous studies have shown that oxidative stress is associated with antibiotic resistance in pathogenic bacteria, and the exposure of bacteria to antibiotics may alter the antioxidant defense system [31, 32]. DOX is a tetracycline antibiotic produced by fermentation of various Streptomyces species [33]. Another study showed that glutathione protects photosystem I from oxidative damage and facilitates antibiotic resistance in cyanobacteria [34].

However, KEGG enrichment analysis showed that AMC-resistant strains were involved in lysine biosynthesis; thyroid hormone synthesis; valine, leucine, and isoleucine degradation; and amino acid and nucleotide sugar metabolism pathways. A previous study has shown that in the β-lactam-generating Actinomycetes, lysine ε-aminotransferase with lysine biosynthesis has been shown to catalyze the first steps of β-lactam antibiotic biosynthesis pathway [35]. It has also been shown that the synthesis of antibiotics heavily depends on α-aminoadipate derived from lysine biosynthesis [36]. Another study showed that the presence of leucine and serine in the growth medium caused partial starvation of isoleucine and valine, resulting in the induction of a strict response and co-resistance to mecillinam in Escherichia coli [37, 38]. Therefore, this study invokes a change of perspective for future research to focus not only on identifying genetic variants underlying the resistance phenotype but also on the regulatory pathways associated with drug resistance.

Altogether, the analysis and prediction of potential mechanisms of the drug resistance in B. pseudomallei will extremely provide the new choice of clinical intervention and improve public health surveillance thus avoiding outbreaks caused by emerging multidrug resistant strains.

Burkholderia pseudomallei is an important pathogen among human which mainly leads to Melioidosi and poses a huge threat to public health. The multi-drug resistance in B. pseudomallei has become a tough problem for prevention and treatment. It has become an urgent issue to investigate the drug resistance features and the mechanisms of its resistance. Hence, the mechanism of antibiotic resistance in B. pseudomallei could provide a perspective for further analysis and potential therapeutic targets for clinical treatment.

Bacterial strains

Clinical samples from patients suspected of having melioidosis were cultured and identified based on biochemical reactions or MALDI-TOF MS. A total of 558 B. pseudomallei isolates were isolated from different patients in 18 cities across Hainan Province from 2003 to 2022 (supplementary Table S1). The culture medium used for bacterial cultivation was blood agar, MacConkey agar, and chocolate agar, and the culture conditions were 35–37 ℃, with or without CO₂.

Genome annotation and pan-genome analysis

Sequenced B. pseudomallei genomes were annotated using Prokka v1.13 [39], followed by pan-genome analysis using Roary v3.13.0, based on the genome annotation files [40]. ABRicate v1.0.1 (https://github.com/tseemann/abricate) was used to search all genes in the pan-genome for drug resistance genes with identity and coverage set to 60%. Annotation information on the drug resistant genes of B. pseudomallei was obtained from ARG-annot [41], CARD [42], MEGARES 2.00 [43], Resfinder, and NCBI AMRFinderPlus [44, 45]. Functional genome annotation was performed using emapper v2.1.7 and the eggNOG database v5.0.2, with default parameters [46, 47]. The core genome alignment generated by Roary was used to construct a phylogenetic tree using FastTree v2.1.10 [48].

Antibiotic resistance tests

According to the requirements of the CLSI M07 [49], BMD panels were prepared by Wenzhou Kont Biology and Technology Co., Ltd. The concentrations of the antimicrobial agents used were as follows: SXT (0.064/1.216-64/1216 µg/mL), ampicillin/clavulanic (0.25/0.125-64/32 µg/mL), MEM (0.064-4 µg/mL), IPM (0.064-4 µg/mL), DOX (0.125-16 µg/mL), CHL (0.5–64 µg/mL), CAZ (0.125-64 µg/mL). Antimicrobial susceptibility testing (AST) was conducted according to the standard procedures outlined in CLSI M07 (1) for aerobic bacteria. Three to five pure colonies were selected to prepare a 0.5 McFarland bacterial suspension using the broth provided. A 60 µL suspension was diluted 200-fold with 12 mL of AST broth, and then 100 µL of the diluted suspension was added to each well. After incubation at 35 ℃ for 16–20 hours, the MIC was manually read and double-checked.

Pan-genome wide association analysis

Pan-GWAS was performed using Scoary v1.6.16 [50] based on AMC- and DOX-resistant phenotypes (interpreted according to the CLSI and EUCAST methods, respectively) and by correcting the population genetic structure based on the phylogenetic tree constructed above to obtain the genes related to the drug resistant phenotypes. The significance of the association was determined according to the adjusted p-value < 0.05 (Benjamini-Hochberg adjustment).

Functional annotation of antibiotic resistant genes

Based on the OR values obtained by pan-GWAS, the genes significantly associated with the drug resistant phenotype were divided into two groups: those that are present in the resistant strains (OR > 1) and those that are absent in the resistant strains (OR < 1). GO and KEGG enrichment analyses were performed using clusterProfiler v3.14.3, with non-unique accessory genes as background. GO terms and KEGG pathways with p-value < 0.05 were determined to be significantly enriched [51, 52].

DOX: doxycycline; AMC: amoxicillin-clavulanic acid; pGWAS: pan-genome-wide association studies; KEGG: Kyoto Encyclopedia of Genes and Genomes; GWAS: Genome-wide association studies; SNPs: single nucleotide polymorphisms; WGS: whole-genome sequencing; CARD: comprehensive antibiotic resistance database; CAZ: ceftazidime; MEM: meropenem; SXT: trimethoprim-sulfamethoxazole; TZP: tazobactam; IPM: imipenem; CHL: chloramphenicol; SCF: cefoperazone-sulbactam; TGC: tigecycline; CIP: ciprofloxacin; OR: odds ratio; GO: Gene Ontology; MALDI-TOF MS: Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Ethics approval and consent to participate

The study was reviewed and approved by the Medical Ethics Committee of Hainan General Hospital (Med-Eth-Re[2023] 305). Informed consent was obtained from the participants. All local, national or international guidelines and legislation were adhered to in the production of this study.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available in the NCBI Bioproject repository.

Competing interests

The authors declare that they have no competing interests

Funding

This research was funded by the Key Research and Development Program of Hainan Province (ZDYF2022SHFZ050), Natural Science Foundation of Hainan (821MS120), and Hainan Provincial Clinical Medical Center.

Authors’ contributions

H.W. conceived and designed the study; M.W. and X.D. prepared experimental samples; M.W., X.D. and X.W. performed the experiments; X.W. and D.H. helped to analyze the data; M.W. and D.H. drew a picture of the article; M.W., X.D. and H.W. wrote the manuscript. H.W. modified the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

Not applicable.

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Genome-wide association study of antibiotic resistance in clinical Burkholderia pseudomallei strains from Hainan province, China

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Abstract

Background

Results

Conclusions

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Background

Results

Accessory genes associated with DOX and AMC resistance

Functional analyses of antibiotic-resistant genes

Discussion

Conclusions

Materials and methods

Bacterial strains

Genome annotation and pan-genome analysis

Antibiotic resistance tests

Pan-genome wide association analysis

Functional annotation of antibiotic resistant genes

Abbreviations

Declarations

References

Tables

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