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Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

ABSTRACT: Multidrug-resistant gram-negative pathogens are a significant health threat. Burkholderia spp. encompass a complex subset of gram-negative bacteria with a wide range of biological functions that include human, animal, and plant pathogens. The treatment of infections caused by Burkholderia spp. is problematic due to their inherent resistance to multiple antibiotics. The major ?-lactam resistance determinant expressed in Burkholderia spp. is a class A ?-lactamase of the PenA family. In this study, significant amino acid sequence heterogeneity was discovered in PenA (37 novel variants) within a panel of 48 different strains of Burkholderia multivorans isolated from individuals with cystic fibrosis. Phylogenetic analysis distributed the 37 variants into 5 groups based on their primary amino acid sequences. Amino acid substitutions were present throughout the entire ?-lactamase and did not congregate to specific regions of the protein. The PenA variants possessed 5 to 17 single amino acid changes. The N189S and S286I substitutions were most prevalent and found in all variants. Due to the sequence heterogeneity in PenA, a highly conserved peptide (18 amino acids) within PenA was chosen as the antigen for polyclonal antibody production in order to measure expression of PenA within the 48 clinical isolates of B. multivorans. Characterization of the anti-PenA peptide antibody, using immunoblotting approaches, exposed several unique features of this antibody (i.e., detected <500 pg of purified PenA, all 37 PenA variants in B. multivorans, and Pen-like ?-lactamases from other species within the Burkholderia cepacia complex). The significant sequence heterogeneity found in PenA may have occurred due to selective pressure (e.g., exposure to antimicrobial therapy) within the host. The contribution of these changes warrants further investigation.

SUBMITTER: Becka SA

PROVIDER: S-EPMC6173980 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

Becka Scott A SA Zeiser Elise T ET Marshall Steven H SH Gatta Julian A JA Nguyen Kevin K Singh Indresh I Greco Chris C Sutton Granger G GG Fouts Derrick E DE LiPuma John J JJ Papp-Wallace Krisztina M KM

Diagnostic microbiology and infectious disease 20180618 3

Multidrug-resistant gram-negative pathogens are a significant health threat. Burkholderia spp. encompass a complex subset of gram-negative bacteria with a wide range of biological functions that include human, animal, and plant pathogens. The treatment of infections caused by Burkholderia spp. is problematic due to their inherent resistance to multiple antibiotics. The major β-lactam resistance determinant expressed in Burkholderia spp. is a class A β-lactamase of the PenA family. In this study, ...[more]

PMID: 29983287

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Project description:BackgroundMelioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei.Methodology/principle findingsHere we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection.Conclusions/significanceIn conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

Project description:Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including β-lactams. β-Lactam resistance in Bcc is conferred largely via PenA-like β-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using β-lactamase inhibitors (β-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M-1 s-1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis-Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M-1 s-1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1's Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl-enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer β-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a β-lactam against B. multivorans producing PenA1 and the R220A variant is promising.

Project description:The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 μg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA β-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA β-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 μg/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ∼65-fold-lower apparent Ki (Kiapp) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (-28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ≥ 512 μg/ml). In conclusion, substitutions in the Ω-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the Ω-loop of PenA of this significant clinical pathogen.

Dataset Information

Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

Publications

Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

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