Exploring Cefiderocol Resistance Mechanisms in Burkholderia pseudomallei.
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ABSTRACT:
SUBMITTER: Hall CM
PROVIDER: S-EPMC10269091 | biostudies-literature | 2023 Jun
REPOSITORIES: biostudies-literature
Publications
Exploring Cefiderocol Resistance Mechanisms in Burkholderia pseudomallei.
Hall Carina M CM Somprasong Nawarat N Hagen Johannah P JP Nottingham Roxanne R Sahl Jason W JW Webb Jessica R JR Mayo Mark M Currie Bart J BJ Podin Yuwana Y Wagner David M DM Keim Paul P Schweizer Herbert P HP
Antimicrobial agents and chemotherapy 20230503 6
Cefiderocol is a siderophore cephalosporin designed mainly for treatment of infections caused by β-lactam and multidrug-resistant Gram-negative bacteria. Burkholderia pseudomallei clinical isolates are usually highly cefiderocol susceptible, with <i>in vitro</i> resistance found in a few isolates. Resistance in clinical B. pseudomallei isolates from Australia is caused by a hitherto uncharacterized mechanism. We show that, like in other Gram-negatives, the PiuA outer membrane receptor plays a ma ...[more]
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Project description:Burkholderia pseudomallei is a gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A ?-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ?256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other ?-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.
| S-EPMC3283585 | biostudies-literature
Project description:Cefiderocol is a cephalosporin designed to treat multidrug-resistant Gram-negative infections. By forming a chelated complex with ferric iron, cefiderocol is transported into the periplasmic space via bacterial iron transport systems and primarily binds to penicillin-binding protein 3 (PBP3) to inhibit peptidoglycan synthesis. This mode of action results in cefiderocol having greater in vitro activity against many Gram-negative bacilli than currently used carbapenems, β-lactam/β-lactamase inhibitor combinations, and cephalosporins. Thus, we investigated the in vitro activity of cefiderocol against a total of 246 clinical isolates of Burkholderia pseudomallei from Queensland, Australia. The collection was composed primarily of bloodstream (56.1%), skin and soft tissue (16.3%), and respiratory (15.9%) isolates. MICs of cefiderocol ranged from ≤0.03 to 16 mg/liter, whereas the MIC90 was 0.125 mg/liter. Based upon CLSI clinical breakpoints for cefiderocol against Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia, three isolates (1.2%) would be classified as nonsusceptible (MIC > 4 mg/liter). Using EUCAST non-species-specific (pharmacokinetic/pharmacodynamic [PK/PD]) clinical breakpoints or those set for Pseudomonas aeruginosa, four isolates (1.6%) would be resistant (MIC > 2 mg/liter). Further testing for coresistance to meropenem, ceftazidime, trimethoprim-sulfamethoxazole, amoxicillin-clavulanate, and doxycycline was performed on the four isolates with elevated cefiderocol MICs (>2 mg/liter); all isolates exhibited resistance to amoxicillin-clavulanic acid, while three isolates also displayed resistance to at least one other antimicrobial. Cefiderocol was found to be highly active in vitro against B. pseudomallei primary clinical isolates. This compound shows great potential for the treatment of melioidosis in countries of endemicity and should be explored further.
| S-EPMC7848980 | biostudies-literature
Project description: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.IMPORTANCEBurkholderia 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.
| S-EPMC5587915 | biostudies-literature
Project description:Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including beta-lactams, aminoglycosides, macrolides, and polymyxins. We used Tn5-OT182 to mutagenize B. pseudomallei to identify the genes involved in aminoglycoside resistance. We report here on the identification of AmrAB-OprA, a multidrug efflux system in B. pseudomallei which is specific for both aminoglycoside and macrolide antibiotics. We isolated two transposon mutants, RM101 and RM102, which had 8- to 128-fold increases in their susceptibilities to the aminoglycosides streptomycin, gentamicin, neomycin, tobramycin, kanamycin, and spectinomycin. In addition, both mutants, in contrast to the parent, were susceptible to the macrolides erythromycin and clarithromycin but not to the lincosamide clindamycin. Sequencing of the DNA flanking the transposon insertions revealed a putative operon consisting of a resistance, nodulation, division-type transporter, a membrane fusion protein, an outer membrane protein, and a divergently transcribed regulatorprotein. Consistent with the presence of an efflux system, both mutants accumulated [3H] dihydro streptomycin, whereas the parent strain did not. We constructed an amr deletion strain, B. pseudomallei DD503, which was hypersusceptible to aminoglycosides and macrolides and which was used successfully in allelic exchange experiments. These results suggest that an efflux system is a major contributor to the inherent high-level aminoglycoside and macrolide resistance found in B. pseudomallei.
| S-EPMC89145 | biostudies-literature
Project description:Much is known about the mode of action of drugs and resistance mechanisms under laboratory growth conditions, but research on the bacterial transcriptional response to drug pressure in vivo or efficacious mode of action and transient resistance mechanisms of clinically employed drugs is limited. Accordingly, to assess active alternative metabolism and transient resistance mechanisms, and identify molecular markers of treatment response, the in vivo transcriptional response of Burkholderia pseudomallei 1026b to treatment with ceftazidime in infected lungs was compared to the in vitro bacterial response in the presence of drug. There were 1,688 transcriptionally active bacterial genes identified that were unique to in vivo treated conditions. Of the in vivo transcriptionally active bacterial genes, 591 (9.4% coding capacity) genes were differentially expressed by ceftazidime treatment. In contrast, only 186 genes (2.7% coding capacity) were differentially responsive to ceftazidime treatment under in vitro culturing conditions. Within the genes identified were alternative PBP proteins that may compensate for target inactivation and transient resistance mechanisms, such as β-lactamses that may influence the potency of ceftazidime. This disparate observation is consistent with the thought that the host environment significantly alters the bacterial metabolic response to drug exposure compared to the response observed under in vitro growth. Notably, this study revealed 184 bacterial genes and ORFs that were unique to in vivo ceftazidime treatment and thus provide candidate molecular markers for treatment response. This is the first report of the unique transcriptional response of B. pseudomallei from host tissues in an animal model of infection and elucidates the in vivo metabolic vulnerabilities, which is important in terms of defining the efficacious mode of action and transient resistance mechanisms of a frontline meliodosis chemotherapeutic, and biomarkers for monitoring treatment outcome.
| S-EPMC5230754 | biostudies-literature
Project description:Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium's intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation ?-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted ?-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine ?-lactams tested and ?16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible P(tac) promoter, increased resistance levels for all ?-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ?85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ?tatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression.
| S-EPMC3129521 | biostudies-literature
Project description:BackgroundBurkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.Principal findingsMicrobiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the 'greasiness' of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.Conclusion/significanceOur results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.
| S-EPMC4006797 | biostudies-literature
Project description: Not available
| S-EPMC4537509 | biostudies-literature
Project description:The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.
| S-EPMC3524147 | biostudies-other
Project description:The objective of this study was to identify putative mechanisms contributing to baseline cefiderocol resistance among carbapenem-resistant Enterobacterales (CRE). We evaluated 56 clinical CRE isolates with no previous exposure to cefiderocol. Cefiderocol and comparator agent minimum inhibitory concentrations (MICs) were determined by broth microdilution. Short-read and/or long-read whole genome sequencing was pursued. Cefiderocol nonwild type (NWT; i.e., MICs ≥4 mg/L) CRE were compared with species-specific reference genomes and with cefiderocol wild type (WT) CRE isolates to identify genes or missense mutations, potentially contributing to elevated cefiderocol MICs. A total of 14 (25%) CRE isolates met cefiderocol NWT criteria. Of the 14 NWT isolates, various β-lactamases (e.g., carbapenemases in Klebsiella pneumoniae and AmpC β-lactamases in Enterobacter cloacae complex) in combination with permeability defects were associated with a ≥ 80% positive predictive value in identifying NWT isolates. Unique mutations in the sensor kinase gene baeS were identified among NWT isolates. Cefiderocol NWT isolates were more likely to be resistant to colistin than WT isolates (29% vs. 0%). Our findings suggest that no consistent antimicrobial resistance markers contribute to baseline cefiderocol resistance in CRE isolates and, rather, cefiderocol resistance results from a combination of heterogeneous mechanisms.
| S-EPMC8885434 | biostudies-literature