Folate pathway inhibitor resistance mechanisms in Burkholderia pseudomallei

2013 Fall.Includes bibliographical references.Antimicrobials are invaluable tools used to facilitate the treatment of infectious diseases. Their use has saved millions of lives since their introduction in the early 1900's. Unfortunately, due to the increased incidence and dispersal of antimicrobial resistance determinants, many of these drugs are no longer efficacious. This greatly limits the options available for treatment of serious bacterial infections, including melioidosis, which is caused by Burkholderia pseudomallei, a Gram-negative saprophyte. This organism is intrinsically resistant to many antimicrobials. Additionally, there have been reports of B. pseudomallei isolates resistant to several of the antimicrobials currently used for treatment, including the trimethoprim and sulfamethoxazole combination, co-trimoxazole. The overarching goal of this project was to identify and characterize mechanisms of trimethoprim and sulfamethoxazole resistance in clinical and environmental isolates, as well as in laboratory induced mutants. Prior to these studies, very little work has been done to identify and characterize the mechanisms by which B. pseudomallei strains are or could become resistant to folate-pathway inhibitors, specifically trimethoprim and sulfamethoxazole. During the initial phases of these studies, we determined the antimicrobial susceptibilities of a large collection of clinical and environmental isolates from Thailand and Australia (n = 65). A high frequency of naturally-occurring resistance to trimethoprim alone (40%) was observed. However these strains were susceptible to sulfamethoxazole and to the co-trimoxazole combination. Trimethoprim resistance in a subset of these strains was due to increased expression of an efflux pump belonging to the resistance nodulation and cellular division (RND) superfamily, BpeEF-OprC, in the presence of trimethoprim. This efflux pump had been previously shown to efflux trimethoprim, chloramphenicol and tetracyclines when expressed in surrogate bacterial strains. The molecular mechanism of increased BpeEF-OprC expression in these isolates remains unknown. Similarly, decreased susceptibility in laboratory mutants selected on trimethoprim were due to mutations leading to amino acid substitutions in BpeT, which caused overexpression of BpeEF-OprC, or FolA, the trimethoprim drug target. This is the first description of mutations to FolA conferring trimethoprim resistance in B. pseudomallei, though similar mutations have been observed in B. cenocepacia and Escherichia coli. A similar study to select for sulfamethoxazole resistance, instead suggested that B. pseudomallei may be able to tolerate high concentrations of the drug. Studies to characterize laboratory induced mutants selected on co-trimoxazole led to the identification of two novel resistance determinants. Mutations to BpeS, a newly named LysR-type regulator with high similarity to the cognate BpeEF-OprC efflux pump regulator, BpeT, cause increased BpeEF-OprC expression in these strains. Additionally mutations to Ptr1, an annotated pteridine reductase, partially contributed to the decreased co-trimoxazole susceptibility. However, it is unclear what function Ptr1 has in the folate synthesis pathway, as deletion of this gene also caused slight decreases in antimicrobial susceptibility. Finally, in a collection of co-trimoxazole resistant clinical isolates from Thailand, high-level expression of the BpeEF-OprC was found in the resistant isolates. A mutation to BpeS was also observed in two of the clinical isolates that had BpeT-independent BpeEF-OprC overexpression. Co-trimoxazole resistant isolates were each resistant to both trimethoprim and sulfamethoxazole individually. However, deletion of the bpeEF-oprC efflux pump structural genes in all isolates resistant to co-trimoxazole or isolates resistant to trimethoprim alone (except those with a mutant FolA) resulted in antimicrobial susceptibility to trimethoprim, co-trimoxazole and sulfamethoxazole. These data suggest that sulfamethoxazole is also a substrate of the BpeEF-OprC efflux pump and this RND pump is the major resistance determinant contributing to clinically relevant folate pathway inhibitor resistance in B. pseudomallei. To summarize, we have identified and described several resistance determinants in B. pseudomallei causing decreased susceptibilities to trimethoprim, sulfamethoxazole and/or co-trimoxazole; these include drug target and metabolic pathway modifications and overexpression of the BpeEF-OprC efflux pump. Further characterization of these mechanisms and the development of specific detection assays could allow for rapid determination of antimicrobial resistance and provide useful information for the development of novel antimicrobials against B. pseudomallei

Evolutionary Instability of Collateral Susceptibility Networks in Ciprofloxacin-Resistant Clinical Escherichia coli Strains

ABSTRACT Collateral sensitivity and resistance occur when resistance development toward one antimicrobial either potentiates or deteriorates the effect of others. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection, and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin-resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression are the main drivers destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks. IMPORTANCE Antimicrobial resistance occurs due to genetic alterations that affect different processes in bacteria. Thus, developing resistance toward one antimicrobial drug may also alter the response toward others (collateral effects). Understanding the mechanisms of such collateral effects may provide clinicians with a framework for informed antimicrobial treatment strategies, limiting the emergence of antimicrobial resistance. However, for clinical implementation, it is important that the collateral effects of resistance development are repeatable and temporarily stable. Here, we show that collateral effects caused by resistance development toward ciprofloxacin in clinical Escherichia coli strains are not temporarily stable because of compensatory mutations restoring the fitness burden of the initial resistance mutations. Consequently, this instability is complicating the general applicability and clinical implementation of collateral effects into treatment strategies

Efflux Pump-mediated Drug Resistance in Burkholderia

Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in B. cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND) family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance

Absence of Bacteria in the Temporal Arteries of Patients with Giant Cell Arteritis (.pdf)

An infectious trigger for giant cell arteritis (GCA) has been considered but no organism has conclusively been linked to GCA. We have previously presented data at NANOS linking GCA to a strain of bacteria. We performed confirmatory experiments to determine if GCA is caused by a bacterial infection. We cultured superficial temporal artery biopsies (STABs) and analyzed the cultures for bacterial growth. We also used 16S rRNA sequencing technology to identify bacterial genomic sequences in STABs

La Charente

19 mai 18811881/05/19 (A10,N4345)-1881/05/19.Appartient à l’ensemble documentaire : PoitouCh

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of Escherichia coli

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses

Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C T ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4) colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen

Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-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 strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect 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 cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes 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 to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is 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 affecting folA, but not folP, and folM, 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.Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research; NIH NIAID [AI065357]; University of Florida Preeminence start-up fundsThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-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 strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect 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 in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium

Average concentration and purity of DNA extraction performed by commercially available DNA extraction kits on <i>Burkholderia pseudomallei</i> spiked whole blood containing EDTA.

<p>Averages based on all blood specimens.</p