Biology subjects

Sarovich, D. S.

Publications and source records attributed to Sarovich, D. S..

16 recordsLinked to original sources

bioRxiv2026

Using population-level whole-genome sequencing to profile colistin resistance evolution dynamics in diverse clinical Pseudomonas aeruginosa lineages

Colistin (CST) is a critical last-line antibiotic for treating multidrug-resistant Pseudomonas aeruginosa infections, especially in chronic respiratory disease. Despite its clinical importance, the P. aeruginosa CST resistome remains incompletely characterised, with most studies focusing on the PAO1 prototypic strain. Here, we used experimental evolution to apply stepwise CST selective pressure to nine P. aeruginosa lineages representing different clinical presentations and diverse genetic backgrounds. Population-level whole-genome sequencing of broth cultures exposed to 2-fold increasing CST (0.0625 to 512 g/mL) was undertaken at multiple concentrations. We identified several known and novel mutational drivers of low- and high-level CST resistance, alongside associated compensatory mutations at allele frequencies (AFs) as low as 5%. Despite diverse clinical and genetic backgrounds, all lineages convergently evolved mutations within the two-component system pmrAB, with most accompanied by phoPQ variants. When canonical TCS remodelling plateaued, populations acquired novel secondary driver mutations, most notably in the lipid A-modifying gene, lpxO2. In addition to AMR driver mutations, strains evolved complex adaptive strategies to mitigate severe CST-induced oxidative and metabolic stress. Adaptations included the emergence of hypermutators and alterations to targeted DNA and protein repair networks. Furthermore, we observed convergent regulatory rewiring of the cyclic-di-GMP network and central metabolism pathways, likely acting as compensatory mechanisms to promote biofilm formation and offset the fitness costs of CST resistance. Using a mixture-aware population genomics approach, we captured intense clonal interference and transient tolerance mutations that failed to reach fixation, illustrating dynamic evolutionary complexities that would be missed with traditional end-point analysis methods.

genomics↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

Prevalence of Pseudomonas aeruginosa in Australian wild birds, native wildlife, livestock and domestic animals

The ESKAPE pathogen, Pseudomonas aeruginosa, poses a serious threat to medical, veterinary, and agricultural practices globally. Understanding P. aeruginosa prevalence in wild bird populations, livestock, and domestic animals is vital for evaluating potential infection reservoirs. In this study, we screened 1,669 DNA samples obtained between 2010 and 2023 from healthy and diseased wild birds (n= 1,101), domestic animals (n= 269), livestock (n= 133), kangaroos (n= 39,) and koalas (n= 127) from Southeast Queensland, Australia, for both P. aeruginosa and overall bacterial load using an ecfX-16S rRNA duplex real-time PCR assay. P. aeruginosa-positive samples were also screened for the two most common fluoroquinolone resistance genotypes, GyrA Thr83Ile and GyrA Asp87Asn. Overall, only 1.8% samples were P. aeruginosa positive, a lower rate than reported in international studies. Livestock samples showed the highest P. aeruginosa prevalence (4.5%, n= 6), primarily horses (7.4%, n= 5), with wild birds (1.5%, n= 17), koalas (1.6% n= 2), and domestic animals (1.9%, n= 5) having the next highest rates. In contrast, no P. aeruginosa positive samples were identified in cattle (n= 45) or kangaroos (n= 39). Nearly all positive wild bird samples originated from eye swabs (94%, n=16). No additional correlation between swab site, health status, or admission cause was identified. The GyrA Thr83Ile variant was seen in 2/30 (6.6%) P. aeruginosa-positive samples, both of horse origin. Our findings provide important insight into the epidemiology of P. aeruginosa in Australian wildlife and domestic animal populations. Further prevalence studies, particularly covering a broader geographical region, are warranted to better elucidate nationwide P. aeruginosa carriage, infection, and fluoroquinolone resistance rates.

microbiology↗

bioRxiv2021

Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.

microbiology↗

bioRxiv2021

Comparative genomics of Nocardia seriolae reveals recent importation and subsequent widespread dissemination in mariculture farms in South Central Coast, Vietnam

Between 2010 and 2015, nocardiosis outbreaks caused by Nocardia seriolae affected many permit farms throughout Vietnam, causing mass fish mortalities. To understand the biology, origin, and epidemiology of these outbreaks, 20 N. seriolae strains collected from farms in four provinces in the South-Central Coast of Vietnam, along with two Taiwanese strains, were analysed using genetics and genomics. Pulsed-field gel electrophoresis identified a single cluster amongst all Vietnamese strains that was distinct from the Taiwanese strains. Like the PFGE findings, phylogenomic and single-nucleotide polymorphism (SNP) genotyping analyses revealed that all Vietnamese N. seriolae strains belonged to a single, unique clade. Strains fell into two subclades that differed by 103 SNPs, with almost no diversity within clades (0-2 SNPs). There was no association between geographic origin and subclade placement, suggesting frequent N. seriolae transmission between Vietnamese mariculture facilities during the outbreaks. Vietnamese strains shared a common ancestor with strains from Japan and China, with the closest strain, UTF1 from Japan, differing by just 217 SNPs from the Vietnamese ancestral node. Draft Vietnamese genomes range from 7.55-7.96 Mbp in size, have an average G+C content of 68.2%, and encode 7,602-7,958 predicted genes. Several putative virulence factors were identified, including genes associated with host cell adhesion, invasion, intracellular survival, antibiotic and toxic compound resistance, and haemolysin biosynthesis. Our findings provide important new insights into N. seriolae epidemiology and pathogenicity and will aid future vaccine development and disease management strategies, with the ultimate goal of nocardiosis-free aquaculture.

genomics↗

bioRxiv2020

Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

SynopsisThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid ([~]40 min) real-time PCR assays targeting the most common extended spectrum {beta}-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY {beta}-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one {beta}-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of {beta}-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

microbiology↗

bioRxiv2020

Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

Several members of the Gram-negative environmental bacterial genus, Achromobacter, are associated with serious infections in immunocompromised individuals, of which Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, comparatively little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management of Achromobacter infections. Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly identify species boundaries, to reassign several incorrectly speciated taxa, and to identify genetic sequences specific for the Achromobacter genus and for A. xylosoxidans. Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans, with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to [~]110 genome equivalents, and detected down to [~]12 and [~]1 genome equivalent/s, respectively. In silico analysis, and laboratory testing of 34 non-Achromobacter isolates and 38 adult CF sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive, and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans, and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

microbiology↗

bioRxiv2019

Comparative genomics confirms a rare melioidosis human-to-human transmission event and reveals incorrect phylogenomic reconstruction due to polyclonality

Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei, is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterise one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs) 259 and 261 were identified in the mothers sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mothers breast milk and the childs cerebrospinal fluid and blood samples contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mothers mixed ST-259/261 sputum sample was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls.\n\nImpact StatementBurkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of high mortality. B. pseudomallei infections occur almost exclusively through contact with contaminated soil and water. Using whole-genome sequencing (WGS), we investigated a rare case of suspected B. pseudomallei transmission from mother to child. The mothers sputum, breast milk and the babys blood and cerebrospinal fluid (CSF) specimens were collected, and DNA was extracted from both pure colonies and primary culture sweeps to capture potential strain mixtures. In-depth analysis of genetic variants identified two strains in the mothers sputum belonging to multilocus sequence types ST-259 and ST-261, whereas the child was infected with only ST-259. Comparative genomics revealed no genetic differences between mother and child ST-259 isolates, providing the strongest possible evidence of transmission to the child via breast milk. The sputum strain mixture was subsequently used to develop a bioinformatic method for identification and quantification of mixtures from WGS data. Using this method, we found ST-259 and ST-261 at an 87%:13% ratio, respectively. Finally, we demonstrate the negative impact that even a single strain mixture event can have on both within-ST and global phylogenomic inferences. Our findings highlight the need for bioinformatic quality control to avoid unintended consequences of phylogenomic incongruence and branch collapse.\n\nData SummaryO_LIWhole-genome sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) and GenBank under BioProject accession number PRJNA559002.\nC_LIO_LIThe GenBank accession number for MSHR0643 assembly is VXLH00000000.1.\nC_LIO_LIThe SRA accession numbers for all raw sequence data are listed in Table 1.\nC_LI\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@c3b580org.highwire.dtl.DTLVardef@c11407org.highwire.dtl.DTLVardef@9e443org.highwire.dtl.DTLVardef@169014borg.highwire.dtl.DTLVardef@d4026c_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary of ST-259 and ST-261 Burkholderia pseudomallei isolates.\n\nC_TABLECAPTION C_TBL RepositoriesAll sequencing data generated as part of this study can be found under the NCBI BioProject PRJNA559002 with accession numbers listed in Table 1.

microbiology↗

bioRxiv2019

Taking the next-gen step: comprehensive antimicrobial resistance detection from Burkholderia pseudomallei genomes

BackgroundAntimicrobial resistance (AMR) poses a major threat to human health. Whole-genome sequencing holds great potential for AMR identification; however, there remain major gaps in comprehensively detecting AMR across the spectrum of AMR-conferring determinants and pathogens. MethodsUsing 16 wild-type Burkholderia pseudomallei and 25 with acquired AMR, we first assessed the performance of existing AMR software (ARIBA and CARD) for detecting clinically relevant AMR in this pathogen. B. pseudomallei was chosen due to limited treatment options, high fatality rate, and AMR caused exclusively by chromosomal mutation (i.e. single-nucleotide polymorphisms [SNPs], insertions-deletions [indels], copy-number variations [CNVs], and functional gene loss). Due to poor performance with existing tools, we developed ARDaP (Antimicrobial Resistance Detection and Prediction) to identify the spectrum of AMR-conferring determinants in B. pseudomallei. ResultsCARD failed to identify any clinically-relevant AMR in B. pseudomallei, ARIBA cannot differentiate AMR determinants from natural genetic variation, and neither CARD or ARIBA can identify CNV or gene loss determinants. In contrast, ARDaP accurately detected all SNP, indel, CNV, and gene loss AMR determinants described in B. pseudomallei (n{approx}50). Additionally, ARDaP accurately predicted three previously undescribed determinants. In mixed strain data, ARDaP identified AMR to as low as ~5% allelic frequency. ConclusionsWe demonstrate that existing AMR software are inadequate for comprehensive AMR detection; ARDaP overcomes the shortcomings of existing tools. Further, ARDaP enables AMR prediction from mixed sequence data down to 5% allelic frequency. ARDaP databases can be constructed for any microbial species of interest for comprehensive AMR detection.

genomics↗

bioRxiv2019

Quantitative real-time PCR assay for the rapid identification of the multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, there is no rapid, cost-effective, and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence may be underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia, with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. genomes identified a 4kb region specific to S. maltophilia, which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive, and cost-effective method for the accurate identification of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.

microbiology↗

bioRxiv2019

Molecular signatures of non-typeable Haemophilus influenzae lung adaptation in paediatric chronic lung disease

Non-typeable Haemophilus influenzae (NTHi), an opportunistic pathogen of the upper airways of healthy children, can infect the lower airways, driving chronic lung disease. However, the molecular basis underpinning NTHi transition from a commensal to a pathogen is not clearly understood. Here, we performed comparative genomic and transcriptomic analyses of 12 paired, isogenic NTHi strains, isolated from the nasopharynx (NP) and bronchoalveolar lavage (BAL) of 11 children with chronic lung disease, to identify convergent molecular signatures associated with lung adaptation. Comparative genomic analyses of the 12 NP-BAL pairs demonstrated that five were genetically identical, with the remaining seven differing by only 1 to 3 mutations. Within-patient transcriptomic analyses identified between 2 and 58 differentially expressed genes in 8 of the 12 NP-BAL pairs, including pairs with no observable genomic changes. Whilst no convergence was observed at the gene level, functional enrichment analysis revealed significant under-representation of differentially expressed genes belonging to Coenzyme metabolism, Function unknown, Translation, ribosomal structure and biogenesis Cluster of Orthologous Groups categories. In contrast, Carbohydrate transport and metabolism, Cell motility and secretion, Intracellular trafficking and secretion, and Energy production categories were over-represented. This observed trend amongst genetically-unrelated NTHi strains provides evidence of convergent transcriptional adaptation of NTHi to paediatric airways that deserves further exploration. Understanding the pathoadaptative mechanisms that NTHi employs to infect and persist in the lower paediatric airways is essential for devising targeted diagnostics and treatments aimed at minimising disease severity, and ultimately, preventing NTHi lung infections and subsequent chronic lung disease in children.

microbiology↗

bioRxiv2019

Tracing the environmental footprint of the Burkholderia pseudomallei lipopolysaccharide genotypes in the tropical \"Top End\" of the Northern Territory, Australia

The Tier 1 select agent Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence in B. pseudomallei include the Burkholderia intracellular motility factor A (bimA) and filamentous hemagglutinin 3 (fhaB3) gene variants. Three lipopolysaccharide (LPS) O-antigen types in B. pseudomallei have been described, which vary in proportion between Australian and Asian isolates. However, it remains unknown if these LPS types can be used as genetic markers for geospatial analysis within a contiguous melioidosis-endemic region. Using a combination of whole-genome sequencing (WGS), statistical analysis and geographical mapping, we examined if the LPS types can be used as geographical markers in the Northern Territory, Australia. The clinical isolates revealed that LPS A prevalence was highest in the Darwin and surrounds (n = 660; 96% being LPS A and 4% LPS B) and LPS B in the Katherine and Katherine remote and East Arnhem regions (n = 79; 60% being LPS A and 40% LPS B). Bivariate logistics regression of 999 clinical B. pseudomallei isolates revealed that the odds of getting a clinical isolate with LPS B was highest in East Arnhem in comparison to Darwin and surrounds (OR 19.5, 95% CI 9.1 - 42.0; p<0.001). This geospatial correlation was subsequently confirmed by geographically mapping the LPS type from 340 environmental Top End strains. We also found that in the Top End, the minority bimA genotype bimABm has a similar remote region geographical footprint to that of LPS B. In addition, correlation of LPS type with multi-locus sequence typing (MLST) was strong, and where multiple LPS types were identified within a single sequence type, WGS confirmed homoplasy of the MLST loci. The clinical, sero-diagnostic and vaccine implications of geographically-based B. pseudomallei LPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.\n\nAuthor SummaryBurkholderia pseudomallei is a pathogenic soil bacterium that causes the disease melioidosis, which occurs in many tropical regions globally and in recent years has emerged in non-tropical regions. Melioidosis has been predicted to affect 165,000 people every year resulting in an estimated 89,000 deaths. Person to person transmission is rare with most cases linked to exposure to the bacterium from the environment. The genetic background of B. pseudomallei has been well studied and variably present genes have been linked to distinct melioidosis disease states and geographic regions, however we still need a stronger understanding of the association of genes with geography. Three lipopolysaccharide types exist in B. pseudomallei and the prevalence of the lipopolysaccharide genes vary between melioidosis endemic regions, but it is unknown if the lipopolysaccharide genes can be used as geographical markers in a single melioidosis-endemic region. In this study, we used a combination of whole-genome sequencing, statistics and geographical mapping to elucidate if the three lipopolysaccharide genes can be used as geographical markers within the Northern Territory, Australia. We show that the three LPS types have distinct but overlapping geographical footprints within a single melioidosis region and can be used as geographic markers alongside a number of other important variably present B. pseudomallei genes.

molecular biology↗

bioRxiv2019

Pathogen to commensal: longitudinal within-host population dynamics, evolution, and adaptation during a chronic >16-year Burkholderia pseudomallei infection

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where the disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this chronic infection to better understand B. pseudomallei persistence in P314s sputum despite intense and repeated therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314s airways over a >16-year period, and also sampled the patients home environment, recovering six closely related B. pseudomallei isolates from water. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a single clonal population. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that, resulting in a highly linear topology. Although these extinctions and persistence could be explained by genetic drift, we observe phenotypic changes consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes changed with time and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression to commensalism by a highly virulent pathogen within a single human host. Author summaryPathogens frequently jump between different hosts and associated adaptation may lead to the emergence of new infectious agents. Such host-jumping evolution is witnessed through endpoint analyses but these cannot capture genetic changes in lineages that have gone extinct. In this study, we have identified and monitored an example of the adaptive evolution of a bacterium often deadly to its mammalian host, in an unprecedented case whereby disease lessened through time and the pathogen adapted to become a part of the commensal human flora. We used genomic analyses to characterize more than 16 years of this evolutionary process and the stepwise mutations that control dictate pathogen interactions with the patient. Soon after infection, mutational changes occurred that allowed the bacterium to remain in the airways without causing disease. This shift towards avirulence was determined based on clinical data and virulence testing in an animal model. In addition, mutations occurred that contributed to the persistence of the bacteria in the patients lungs. Finally, we found evidence for the evolutionary emergence and persistence of two distinct lineages of the bacterium over the last 13 years, presenting interesting questions about niche utilization and separation. Bacteria are ubiquitous in the human body and almost all are beneficial or benign. In this study, we document the evolutionary conversion of a normally deadly bacterium into a commensal.

evolutionary biology↗

bioRxiv2018

Development and validation of a triplex qPCR assay to detect efflux pump-mediated antibiotic resistance in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the deadly tropical disease melioidosis, is intrinsically resistant to many antibiotics, leaving few effective treatment options. Trimethoprim-sulfamethoxazole (SXT), meropenem (MEM) and doxycycline (DOX) are valuable antibiotics for melioidosis treatment due to inherently low or no primary resistance. Although considered rare, upregulation of one or more resistance-nodulation-division (RND) efflux pumps is now known to lead to acquired resistance towards these drugs in B. pseudomallei. Here, we developed a triplex quantitative PCR assay to detect upregulation of the three clinically relevant RND efflux systems: AmrAB-OprA, BpeB-OprB and BpeEF-OprC. The triplex assay was tested on seven clinically-derived B. pseudomallei isogenic pairs, where the latter strain of each pair had altered regulator activity and exhibited reduced susceptibility to SXT, MEM or DOX. The triplex assay accurately detected efflux pump upregulation between isogenic pairs, which corresponded with decreased antibiotic susceptibility. We further verified assay performance on eight laboratory-generated B. pseudomallei mutants encoding efflux pump regulator mutations. Targeting antibiotic resistance in B. pseudomallei using molecular genotyping provides clinicians with a rapid tool to identify potential treatment failure in near real-time, enabling informed alteration of treatment during an infection and improved patient outcomes.\n\nIMPORTANCEThe melioidosis bacterium Burkholderia pseudomallei is intrinsically resistant to many antibiotics, limiting treatment options to a handful of drugs including meropenem, doxycycline and trimethoprim-sulfamethoxazole. Although rare, there have now been several documented melioidosis cases where resistance to these antibiotics has developed during an infection, leading to treatment failure and increased mortality rates. Interestingly, all strains resistant to these drugs exhibit increased efflux pump expression, representing a shared molecular signature that can be exploited for rapid diagnostic purposes. Here, we developed and validated a single-tube real-time qPCR assay to detect clinically relevant efflux pump upregulation in B. pseudomallei, an important first step towards high-level resistance. This triplex assay offers a drastically reduced turn-around-time compared to current methodology, enabling earlier detection of resistance emergence. Implementation of this new diagnostic will aid clinicians in the selection of appropriate therapy, thereby minimizing resistance development and treatment failure for this high-mortality disease.

microbiology↗

bioRxiv2017

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognized as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between four and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between patient CF9 strains, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harbored duplications that concomitantly increased expression of the {beta}-lactamase gene penA, and a 35kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the antibiotic resistance-nodulation-division (RND) efflux pump BpeEF-OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically-adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

evolutionary biology↗

bioRxiv2017

Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a Gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug, meropenem, is the current gold-standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance towards meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Here, we document 11 melioidosis cases in which B. pseudomallei isolates developed decreased susceptibility towards meropenem during treatment, including two cases not treated with this antibiotic. Meropenem minimum inhibitory concentrations increased over time from 0.5-0.75 to 3-8 g/mL. Using comparative genomics, we identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, leading to over-expression of their corresponding pumps. The most commonly affected pump was AmrAB-OprA, although alterations in the local regulators of BpeEF-OprC or BpeAB-OprB were observed in three cases. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. Further, we document two concerning examples of severe melioidosis where the reduced treatment efficacy of meropenem was associated with a fatal outcome.\n\nSignificance StatementThe bacterium Burkholderia pseudomallei, which causes the often-fatal tropical disease melioidosis, is difficult to eradicate. Due to high levels of intrinsic antibiotic resistance, only a handful of antibiotics are effective against this pathogen. One of these, meropenem, is commonly used in the treatment of melioidosis patients who are unresponsive to other treatments or are critically ill. Here, we describe 11 melioidosis cases whereby patients exhibited prolonged or repeated infections that were associated with the development of decreased meropenem susceptibility. We identified the molecular basis for this decreased susceptibility in latter B. pseudomallei isolates obtained from these patients, and functionally confirmed the mechanism conferring this phenotype. Our findings have important ramifications for the diagnosis, treatment and management of life-threatening melioidosis cases.

microbiology↗