scholarly journals The BpeEF-OprC Efflux Pump Is Responsible for Widespread Trimethoprim Resistance in Clinical and Environmental Burkholderia pseudomallei Isolates

2013 ◽  
Vol 57 (9) ◽  
pp. 4381-4386 ◽  
Author(s):  
Nicole L. Podnecky ◽  
Vanaporn Wuthiekanun ◽  
Sharon J. Peacock ◽  
Herbert P. Schweizer
Keyword(s):  
Dihydrofolate Reductase ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Eradication Therapy ◽  
Northern Australia ◽  
Northeast Thailand ◽  
Environmental Isolates ◽  
Transcriptional Terminator ◽  
Content Type ◽  
Primary Drug

ABSTRACTTrimethoprim-sulfamethoxazole (co-trimoxazole) is the primary drug used for oral eradication therapy ofBurkholderia pseudomalleiinfections (melioidosis). Here, we demonstrate that trimethoprim resistance is widespread in clinical and environmental isolates from northeast Thailand and northern Australia. This resistance was shown to be due to BpeEF-OprC efflux pump expression. No dihydrofolate reductase target mutations were involved, although frequent insertion of ISBma2 was noted within the putativefolAtranscriptional terminator. All isolates tested remained susceptible to trimethoprim-sulfamethoxazole, suggesting that resistance to trimethoprim alone in these strains probably does not affect the efficacy of co-trimoxazole therapy.

scholarly journals Distribution of Burkholderia pseudomallei in Northern Australia, a Land of Diversity

2014 ◽  
Vol 80 (11) ◽  
pp. 3463-3468 ◽  
Author(s):  
Evan McRobb ◽  
Mirjam Kaestli ◽  
Erin P. Price ◽  
Derek S. Sarovich ◽  
Mark Mayo ◽  
...  
Keyword(s):  
Population Structure ◽  
Burkholderia Pseudomallei ◽  
Northern Region ◽  
Northern Australia ◽  
Source Attribution ◽  
Environmental Isolates ◽  
Sampling Area ◽  
Dissimilarity Matrix ◽  
Content Type ◽  
Fragmented Habitats

ABSTRACTBurkholderia pseudomalleiis a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 AustralianB. pseudomalleistrains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found twoB. pseudomalleipopulations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming thatB. pseudomalleiis widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed thatB. pseudomalleipopulations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting thatB. pseudomalleipopulations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturableB. pseudomalleiisolates to cause disease. Our findings have important implications for understanding the ecology ofB. pseudomalleiin Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis.

scholarly journals Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Julia V. Bugrysheva ◽  
David Sue ◽  
Jay E. Gee ◽  
Mindy G. Elrod ◽  
Alex R. Hoffmaster ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Clavulanic Acid ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Point Mutations ◽  
Comparative Genomic ◽  
Content Type ◽  
Reductase Gene ◽  
Amoxicillin Clavulanic Acid ◽  
Imipenem Resistance

ABSTRACT Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and β-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A β-lactamase and was previously implicated in resistance to β-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

scholarly journals Mechanisms of Resistance to Folate Pathway Inhibitors inBurkholderia pseudomallei: Deviation from the Norm

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Nicole L. Podnecky ◽  
Katherine A. Rhodes ◽  
Takehiko Mima ◽  
Heather R. Drew ◽  
Sunisa Chirakul ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Burkholderia Pseudomallei ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Oral Drug ◽  
Folate Pathway ◽  
Carboxy Terminal ◽  
Effector Binding ◽  
Terminal Effector

ABSTRACTThe trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment ofBurkholderia pseudomalleiinfections. Previous studies demonstrated that theB. pseudomalleiBpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strainB. pseudomalleiBp82, we now show thatin vitroacquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due tobpeTmutations or by BpeEF-OprC overexpression due tobpeSmutations. Mutations inbpeTaffect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations infolM, encoding a pterin reductase. Genetic analyses of these mutants established that bothbpeSmutations andfolMmutations contribute to co-trimoxazole resistance, although the exact role offolMremains to be determined. Mutations affectingbpeT,bpeS, andfolMare common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance inB. pseudomalleiis a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.IMPORTANCEBurkholderia pseudomalleicauses melioidosis, a tropical disease that is difficult to treat. The bacterium’s resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due tofolAandfolPmutations does occur. Co-trimoxazole resistance inB. pseudomalleiis rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affectingfolA, but notfolP, andfolM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

scholarly journals Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

2013 ◽  
Vol 81 (4) ◽  
pp. 1121-1128 ◽  
Author(s):  
Cristine G. Campos ◽  
Luke Borst ◽  
Peggy A. Cotter
Keyword(s):  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Lung Epithelial Cells ◽  
Wild Type ◽  
Passenger Domain ◽  
Content Type ◽  
Reading Frame ◽  
Type V

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) inB. pseudomalleistrain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ tobcaAis Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations ofbcaAandbcaBwere constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaAand Bp340ΔbcaBmutants to wild-typeB. pseudomalleiin vitrodemonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

scholarly journals Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Jessica R. Webb ◽  
Erin P. Price ◽  
Bart J. Currie ◽  
Derek S. Sarovich
Keyword(s):  
Burkholderia Pseudomallei ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Intrinsic Resistance ◽  
Resistance Development ◽  
Content Type ◽  
Treatment Regimens ◽  
Resistance Nodulation Division ◽  
Lengthy Treatment ◽  
Prolonged Antibiotic Therapy

ABSTRACT The soil-dwelling bacterium Burkholderia pseudomallei is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward B. pseudomallei means that melioidosis treatment relies on prolonged antibiotic therapy, which can last up to 6 months in duration or longer. Due to intrinsic resistance, few antibiotics are effective against B. pseudomallei. The lengthy treatment regimen required increases the likelihood of resistance development, with subsequent potentially fatal relapse. Doxycycline (DOX) has historically played an important role in the eradication phase of melioidosis treatment. Both primary and acquired DOX resistances have been documented in B. pseudomallei; however, the molecular mechanisms underpinning DOX resistance have remained elusive. Here, we identify and functionally characterize the molecular mechanisms conferring acquired DOX resistance in an isogenic B. pseudomallei pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative S-adenosyl-l-methionine-dependent methyltransferase (encoded by BPSL3085), which we propose leads to altered ribosomal methylation, thereby decreasing DOX binding efficiency. The second mutation altered the function of the efflux pump repressor gene, amrR, resulting in increased expression of the resistance-nodulation-division efflux pump, AmrAB-OprA. Our findings highlight the diverse mechanisms by which B. pseudomallei can become resistant to antibiotics used in melioidosis therapy and the need for resistance monitoring during treatment regimens, especially in patients with prolonged or recrudesced positive cultures for B. pseudomallei.

scholarly journals Burkholderia pseudomallei Penetrates the Brain via Destruction of the Olfactory and Trigeminal Nerves: Implications for the Pathogenesis of Neurological Melioidosis

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
James A. St. John ◽  
Jenny A. K. Ekberg ◽  
Samantha J. Dando ◽  
Adrian C. B. Meedeniya ◽  
Rachel E. Horton ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Nasal Cavity ◽  
Burkholderia Pseudomallei ◽  
Basal Layer ◽  
The Body ◽  
Northern Australia ◽  
Cranial Cavity ◽  
Content Type ◽  
Trigeminal Nerves ◽  
The Brain

ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.

scholarly journals First Genome Sequence of a Burkholderia pseudomallei Isolate in China, Strain BPC006, Obtained from a Melioidosis Patient in Hainan

2012 ◽  
Vol 194 (23) ◽  
pp. 6604-6605 ◽  
Author(s):  
Yao Fang ◽  
Yong Huang ◽  
Qian Li ◽  
Hai Chen ◽  
Zhen Yao ◽  
...  
Keyword(s):  
Antibiotic Therapy ◽  
Southeast Asia ◽  
Genome Sequence ◽  
High Mortality ◽  
Burkholderia Pseudomallei ◽  
Northern Australia ◽  
Content Type ◽  
Relapse Rates

ABSTRACTMelioidosis, caused byBurkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence ofBurkholderia pseudomalleistrain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.

scholarly journals Surprisingly Low Seroprevalence of Burkholderia pseudomallei in Exposed Healthy Adults in the Darwin Region of Tropical Australia Where Melioidosis Is Highly Endemic

2013 ◽  
Vol 20 (5) ◽  
pp. 759-760 ◽  
Author(s):  
Gemma L. James ◽  
Ben Delaney ◽  
Linda Ward ◽  
Kevin Freeman ◽  
Mark Mayo ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Healthy Adults ◽  
Northeast Thailand ◽  
Hemagglutination Assay ◽  
Seropositivity Rate ◽  
Content Type ◽  
Indirect Hemagglutination ◽  
Tropical Australia

ABSTRACTIn the Darwin region of Australia where melioidosis is highly endemic, only 11/354 (3%) healthy residents were seropositive by indirect hemagglutination assay, despite extensive exposure toBurkholderia pseudomallei. None developed melioidosis, but some described a prior self-limiting illness. This seropositivity rate is much lower than that seen in northeast Thailand, where melioidosis is similarly highly endemic, potentially reflecting important differences between these two locations in the epidemiology of melioidosis.

scholarly journals Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics

2015 ◽  
Vol 82 (3) ◽  
pp. 954-963 ◽  
Author(s):  
Erin P. Price ◽  
Derek S. Sarovich ◽  
Emma J. Smith ◽  
Barbara MacHunter ◽  
Glenda Harrington ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Large Scale ◽  
Phylogenetic Reconstruction ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Antibiotic Administration ◽  
Northern Australia ◽  
Content Type ◽  
Clone Sequence ◽  
Asian Isolate

ABSTRACTMelioidosis is a disease of humans and animals that is caused by the saprophytic bacteriumBurkholderia pseudomallei. Once thought to be confined to certain locations, the known presence ofB. pseudomalleiis expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction ofB. pseudomalleipopulations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455B. pseudomalleigenomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmentalB. pseudomalleiisolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.

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