Biology subjects

Bonomo, R. A.

Publications and source records attributed to Bonomo, R. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Adaptive landscape of the metallo-β-lactamase NDM to emerging β-lactam-based therapies

The New Delhi metallo-{beta}-lactamase (NDM) is a major determinant of carbapenem resistance. This has prompted the development of novel therapeutic strategies to treat NDM producers. Since these therapies impose new selective pressures, their clinical deployment may favor NDM variants carrying escape mutations. To anticipate these events and inform future therapeutic decisions, we explored the evolutionary landscape of NDM-1 under different selective constraints. To this end, we generated a highly diverse library of blaNDM variants and challenged it with zinc starvation, antibiotics or {beta}-lactam/{beta}-lactamase inhibitor combinations that represent novel and emerging therapies. Selection under zinc limitation and cefotaxime identified mutational trajectories that recapitulate clinical NDM evolution, validating the diversity of the library and its predictive nature. Drug-specific selections revealed sharply different evolutionary pathways. Mecillinam selected a narrow evolutionary pathway centered on residues N220 and M67 which enhance productive active-site interactions with this penicillin. Cefepime/taniborbactam selected multiple escape routes, dominated by substitutions at E152 and, secondarily, K211, that impair productive interaction with taniborbactam. In contrast, cefiderocol/xeruborbactam and aztreonam/avibactam failed to select NDM variants conferring an improved resistance phenotype. These results show that NDM evolution is constrained by the chemistry of each therapeutic challenge. Substrate adaptation is possible for mecillinam, inhibitor escape is readily accessible for taniborbactam, whereas aztreonam- and xeruborbactam-based strategies impose high evolutionary barriers on NDM. Mapping drug-specific evolutionary landscapes can help anticipate resistance before clinical deployment and prioritize therapeutic strategies less likely to drive NDM-mediated escape. ImportanceCarbapenem-resistant infections by Enterobacterales are increasingly difficult to treat because bacteria can destroy some of the most powerful antibiotics used in the clinic. This study focuses on Escherichia coli carrying variants of the New Delhi metallo-{beta}-lactamase, an enzyme that enables bacteria to resist carbapenem antibiotics. New therapies are being developed to overcome this problem, but bacteria may evolve again when exposed to these treatments. Here, we tested how New Delhi metallo-{beta}-lactamase can adapt under the evolutionary pressure of last-resort therapies. The results show that not all treatments carry the same evolutionary risk. Some drugs allow the enzyme to adapt through specific mutations, whereas other drug combinations make such escape much harder. This work helps predict which treatments are more likely to remain effective and which may more readily select resistance. This information can guide the design and use of future therapies against resistant bacterial infections.

microbiology↗

bioRxiv2025

A Mechanism Based Pharmacokinetic/Pharmacodynamic Analysis of Polymyxin B-Based Combination Therapy Against Carbapenem-Resistant Klebsiella pneumoniae Isolates with Diverse Phenotypic and Genotypic Resistance Mechanisms

Increased resistance to {beta}-lactams/{beta}-lactamase inhibitor by mutations in {beta}-lactamases gene, porin mutations and efflux pumps complicates the management of carbapenem-resistant Klebsiella pneumoniae (CRKP). Polymyxin B (PMB) based combination therapy is considered as a best alternative treatment for those middle and low-income countries that cannot access to the latest medicines. Its crucial to know both phenotypic and genotypic characteristics of a pathogen to understand the killing effect of each drug and its combinations. Hence, our objective of this study was to incorporate mechanistic insights gained from resistance mechanisms to develop a mechanism based pharmacokinetic/pharmacodynamic model (MBM). Six clinical CRKP isolates were used for static concentration time kill (SCTK) assays to evaluate the rate and extent of killing by monotherapy, double and triple combinations using PMB, meropenem and fosfomycin. A MBM was developed using the SCTK data in S-ADAPT. The MBM estimated lower maximum killing rate constant of PMB (3.61 h-{superscript 1}) in an isolate with non-functional MgrB and high-level phenotypic resistance. Based upon model discrimination and PMBs outer membrane disruption, mechanistic synergy was included in 3 isolates which has porin mutations. Mechanistic synergy of PMB was 83-88% with meropenem and 81-98% with fosfomycin. The PMB concentration required to achieve 50% of synergy was 0.48-0.64 mg/L. Simulations with a lower PMB regimen (1mg/kg q12h) and fosfomycin (8g q8h) showed >73% reduction in area under the bacterial load-versus-time curve for four isolates. The triple combination showed 67.7% reduction in non-carbapenamase producing isolate. This study demonstrates that a low dosing regimen of PMB can produce synergistic effects in combination therapy and might be effective in managing infections caused by CRKP, including PMB resistant isolates. Author summaryAntimicrobial resistance is a major concern in treating infectious diseases. CRKP bacterial isolates are resistant to most of the novel antimicrobial agents. One of the primary resistant mechanisms is restricting the permeability of drugs to the site of action. Polymyxin B, an antimicrobial agent, disrupts the bacterial outer membrane, enhancing the permeability of other drugs. When using combination therapy with polymyxin B, selecting drugs with different mechanisms of action is crucial to enhance synergistic effects and improve treatment efficacy. In our study we chose to evaluate the efficacy of meropenem and fosfomycin in double and triple combination therapies. Mechanism based models (MBMs) are the strongest tool to analyse the time course bacterial load data. Our study provides insights into applying available phenotypic and genotypic information to refine and enhance the accuracy of MBMs. Final model simulations revealed that low exposure of polymyxin B below the nephrotoxic threshold was sufficient to produce synergistic effects when combined with fosfomycin and meropenem. Additionally, we found that polymyxin B combination with fosfomycin was more effective compared to meropenem in treating CRKP.

pharmacology and toxicology↗

bioRxiv2025

Emerging Resistance to Novel -βLactam β-Lactamase Inhibitor Combinations in Klebsiella pneumoniae bearing KPC Variants

BackgroundKlebsiella pneumoniae carbapenemase (KPC) variants, predominantly KPC-2 and KPC-3, are significant global resistance mechanisms. KPC-2 and KPC-3 confer resistance to a broad range of {beta}-lactams, including carbapenems, while remaining susceptible to ceftazidime-avibactam (CZA). Recently, new KPC variants have developed resistance to CZA through mutations, insertions, or deletions in regions such as the {Omega}-loop, 240-loop (237-243 aa), and 270-loop (266-275 aa). This study aimed to investigate the collateral resistance to cefiderocol (FDC) and cefepime/zidebactam (FPZ) among isolates with these mutations. MethodsFifteen clinical isolates of KPC-producing Klebsiella spp. were analyzed, representing 15 distinct variants. Antimicrobial susceptibility testing determined the MICs for CZA, carbapenems, FDC, FPZ, and other antibiotics. Synergy between CZA and FDC was assessed. Whole-genome sequencing (WGS) was used to identify mutations contributing to resistance. ResultsCZA resistance was confirmed in 12 of the 15 KPC variants. Collateral resistance to FDC was observed in eight isolates, with five exhibiting spontaneous resistant subpopulations. Six FDC-resistant strains had mutations in the 270-loop (266-275 aa). Collateral resistance to FPZ was seen in three KPC variants, especially those with mutations in the 270-loop (266-275 aa), though many {Omega}-loop and 240-loop (237-243 aa) mutants remained susceptible. WGS of FDC-resistant subpopulations revealed additional mutations in ompC, rpoC, dksA, and cirA. ConclusionsThis study demonstrates that emerging KPC variants showing resistance to CZA also exhibit resistance to FDC, with collateral resistance to FPZ observed to a lesser extent. Identifying mutations in blaKPC, cirA, and other genes is important to understand resistance mechanisms for effective therapies.

microbiology↗

bioRxiv2025

Mechanistic Basis for Inhibition of the Extended Spectrum Class A b-Lactamase GES-1 by Tazobactam and Enmetazobactam

Expression of {beta}-lactamases is the primary form of {beta}-lactam antibiotic resistance in Gram-negative bacteria. Enmetazobactam is a penicillanic acid sulfone (PAS) that inhibits extended spectrum {beta}-lactamases (ESBLs) by forming an acyl-enzyme complex that eventually breaks down to an irreversible lysinoalanine crosslink. In contrast, enmetazobactam inhibits the class A carbapenemase KPC-2 via an acyl-enzyme that does not lead to lysinoalanine crosslink formation. This difference correlates with greater inhibitory potency of enmetazobactam against class A ESBLs, compared to carbapenemases. The GES enzymes, unlike other class A {beta}-lactamase families, show progression from carbapenem-inhibited to carbapenem-hydrolysing phenotypes through single point mutations. We present crystal structures of GES-1, a globally disseminated ESBL, as the enmetazobactam- and tazobactam-derived acyl-enzymes. The complexes differ in the identities of their respective covalent adducts, with the catalytic Ser70 acylated by a 214 Da enmetazobactam-derived fragment, whereas tazobactam has fragmented to a 70 Da aldehyde. The tautomeric form of the enmetazobactam-derived ligand is verified by high-level QM/MM calculations, revealing the trans-enamine as the most thermodynamically stable tautomer, that adopts an optimised conformation that best matches the experimentally observed electron density appended to the side chain oxygen of Ser70. In contrast to previous findings for the ESBL CTX-M-15, mass spectrometry provides no evidence for lysinoalanine crosslink formation on reaction of GES-1 with (enme)tazobactam, providing further evidence that PAS inhibitors inhibit different class A {beta}-lactamases by different mechanisms. This work reveals new details of the basis for PAS inhibition of diverse {beta}-lactamases, and will guide development of future {beta}-lactamase inhibitors.

biochemistry↗

bioRxiv2024

OXA beta-lactamases from Acinetobacter spp. are membrane-bound and secreted into outer membrane vesicles

{beta}-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced {beta}-lactamases was performed that revealed a predominance of putative lipidated enzymes in the class D OXAs. Namely, 60% of the OXA class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with {beta}-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (> 99%) putative lipidated OXAs are present in Acinetobacter spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in Acinetobacter baumannii. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from Acinetobacter baumannii cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect A. baumannii, Escherichia coli and Pseudomonas aeruginosa cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound {beta}-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated {beta}-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which Acinetobacter spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them. IMPORTANCE{beta}-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving {beta}-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated {beta}-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of class D OXA {beta}-lactamases (all from Acinetobacter spp) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40 are membrane-bound proteins in A. baumannii. This cellular localization favors secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. {beta}-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of {beta}-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by Acinetobacter spp., particularly in polymicrobial infections, being a potent evolutionary advantage.

microbiology↗

bioRxiv2024

Dynamic Differences in Clinically relevant Pen β-Lactamases from Burkholderia spp.

Antimicrobial resistance (AMR) is a global threat, with Burkholderia species contributing significantly to difficult-to-treat infections. Ambler Class A Pen {beta}-lactamases are produced by all Burkholderia spp., and their mutation or overproduction leads to the resistance of {beta}-lactam antibiotics. This study investigates the dynamic differences among four Pen {beta}-lactamases (PenA, PenI, PenL and PenP) using machine learning driven enhanced sampling molecular dynamics simulations, Markov State Models (MSMs), convolutional variational autoencoder-based deep learning (CVAE) and the BindSiteS-CNN model. Although sharing the same catalytic mechanisms, these enzymes exhibit distinct dynamic features due low sequence identity, resulting in different substrate profiles and catalytic turnover. The BindSiteS-CNN model further reveals local active site dynamics, offering insights into the Pen {beta}-lactamase evolutionary adaptation. Our findings reported here identify critical mutations and proposes new hotspots affecting Pen {beta}-lactamase flexibility and function, which can be used to fight emerging resistance in these enzymes.

biophysics↗

bioRxiv2024

Functionally Important Residues from Graph Analysis of Coevolved Dynamic couplings

The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine tune protein dynamics through compensatory mutations, either to improve the proteins performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics (MD) simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue-residue interactions, identifies communities of key residue groups and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings--residue pairs with critical dynamical interactions that have been preserved during evolution--DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded {beta}-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.

evolutionary biology↗

bioRxiv2024

Development of non-β-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to {beta}-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC50: >78M), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC50 {approx} 1M), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (SNAr) reaction, as well as its association with the closure of the major active site of PBP2a.

microbiology↗

bioRxiv2024

Hetero-antagonism of avibactam and sulbactam with cefiderocol in carbapenem-resistant Acinetobacter spp.

The emergence of Gram-negative bacteria resistant to multiple antibiotics, particularly carbapenem-resistant (CR) Acinetobacter strains, poses a significant threat globally. Despite efforts to develop new antimicrobial therapies, limited progress has been made, with only two drugs--cefiderocol and sulbactam-durlobactam--showing promise for CR-Acinetobacter infections. Cefiderocol, a siderophore cephalosporin, demonstrates promising efficacy in the treatment of Gram-negative infections. However, resistance to cefiderocol has been reported in A. baumannii. Combination therapies, such as cefiderocol with avibactam or sulbactam, show reduced MICs against cefiderocol-non-susceptible strains with in vivo efficacy, although the outcomes can be complex and species-specific. In the present work, the molecular characterization of spontaneous cefiderocol-resistant variants, a CRAB strain displaying antagonism with sulbactam and an A. lwoffii strain showing antagonism with avibactam, were studied. The results reveal intriguing insights into the underlying mechanisms, including mutations affecting efflux pumps, transcriptional regulators, and iron homeostasis genes. Moreover, gene expression analysis reveals significant alterations in outer membrane proteins, iron homeostasis, and {beta}-lactamases, suggesting adaptive responses to selective pressure. Understanding these mechanisms is crucial for optimizing treatment strategies and preventing adverse clinical outcomes. This study highlights the importance of preemptively assessing drug synergies to navigate the challenges posed by antimicrobial resistance in CR-Acinetobacter infections.

microbiology↗

bioRxiv2024

{Omega}-Loop mutations control the dynamics of the active site by modulating a network of hydrogen bonds in PDC-3 β-lactamase

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. Using reinforcement learning-driven molecular dynamics simulations and constant pH MD, we investigate how clinically observed mutations in the {Omega}-loop (at residues V211, G214, E219, and Y221) alter the structure and function of PDC-3. Our findings reveal that these substitutions modulate the dynamic flexibility of the {Omega}-loop and the R2-loop, reshaping the cavity of the active site. In particular, E219K and Y221A disrupt the tridentate hydrogen bond network around K67, thus lowering its pKa and promoting proton transfer to the catalytic residue S64. Markov state models reveal that E219K achieves enhanced catalysis by adopting stable, long-lived active conformations, whereas Y221A facilitates activity by rapidly toggling between bond-formed and bond-broken states. In addition, substitutions influence key hydrogen bonds that control the opening and closure of the active-site pocket, consequently influencing the overall size. The pocket expands in all nine clinically identified variants, creating additional space to accommodate bulkier R1 and R2 cephalosporin side chains. Taken together, these results provide a mechanistic basis for how single residue substitutions in the {Omega}-loop affect catalytic activity. Insights into the structural dynamics of the catalytic site advance our understanding of emerging {beta}-lactamase variants and can inform the rational design of novel inhibitors to combat drug-resistant P. aeruginosa.

biophysics↗

bioRxiv2024

Deciphering the co-evolutionary dynamics of L2 β-lactamases via Deep learning

L2 {beta}-lactamases, a serine-based class A {beta}-lactamases expressed by Stenotrophomonas maltophilia plays a pivotal role in antimicrobial resistance. However, limited studies have been conducted on these important enzymes. To understand the co-evolutionary dynamics of L2 {beta}-lactamase, innovative computational methodologies, including adaptive sampling molecular dynamics simulations, and deep learning methods (convolutional variational autoencoders and BindSiteS-CNN) explored conformational changes and correlations within the L2 {beta}-lactamase family together with other representative class A enzymes including SME-1 and KPC-2. This work also investigated the potential role of hydrophobic nodes and binding site residues in facilitating the functional mechanisms. The convergence of analytical approaches utilized in this effort yielded comprehensive insights into the dynamic behaviour of the {beta}-lactamases, specifically from an evolutionary standpoint. In addition, this analysis presents a promising approach for understanding how the class A {beta}-lactamases evolve in response to environmental pressure and establishes a theoretical foundation for forthcoming endeavours in drug development aimed at combating antimicrobial resistance. SynopsisDeep learning is used to reveal the dynamic co-evolutionary patterns of L2 {beta}-lactamases. O_LIAnalysis of hydrophobic nodes and binding site residues provides a detailed understanding of both local and global dynamic evolution, which explain the functional divergences. C_LIO_LIThe employment of two distinct deep learning models, the Convolutional Variational Autoencoder (CVAE) and BindSiteS-CNN, facilitates the investigation of conformational shifts, thereby depicting the dynamic evolution of L2 {beta}-lactamases. C_LIO_LIThe effectiveness of CVAE and BindSiteS-CNN in dynamic classification is corroborated with selected features. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/575584v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1db15forg.highwire.dtl.DTLVardef@168043eorg.highwire.dtl.DTLVardef@1eebb96org.highwire.dtl.DTLVardef@5d8019_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Synergistic Effects of Sulopenem in Combination with Cefuroxime or Durlobactam against Mycobacterium abscessus

Mycobacterium abscessus (Mab) affects patients with immunosuppression, Cystic Fibrosis (CF), or underlying structural lung diseases. Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual {beta}-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with a {beta}-lactamase inhibitors [BLI; avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration MIC values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 [≤] 0.25 g/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding [~]1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in additional reduction of CFUs ([~]3 Log10 for sulopenem-CXM and AVI and [~]4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (LDTs; LDTMab1-LDTMab4), Penicillin-Binding-Protein B (PBP-B), and D,D-Carboxypeptidase (DDC). Acyl complexes identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B, and CXM with LdtMab2 and PBP-B. Molecular docking suggested formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the {beta}-lactam carbonyl carbon, leading to the cleavage of the {beta}-lactam ring, and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLI. These findings potentially broaden selection of oral therapeutic agents to combat Mab.

microbiology↗

bioRxiv2023

The evolution of diverse antimicrobial responses in vancomycin-intermediate Staphylococcus aureus and its therapeutic implications

Staphylococcus aureus bacteremia is typically treated empirically with vancomycin, with therapy later tailored based on susceptibility results. However, these tests occur before vancomycin exposure and do not account for adaptation during empiric treatment that can alter S. aureus susceptibility to first-line drugs. To investigate these collateral drug responses, we experimentally evolved 18 methicillin-susceptible S. aureus (MSSA) populations under increasing vancomycin concentrations until they achieved intermediate resistance. Genomic sequencing revealed two distinct adaptive pathways characterized by mutations in the WalKR regulon, affecting cell wall metabolism, or rpsU, impacting translational stress responses. These pathways correlated with divergent collateral sensitivity profiles to first-line antibiotics. By developing a Collateral Response Score (CRS), we quantified the probability and magnitude of these responses, demonstrating that evolutionary dynamics critically influence resistance outcomes. Our findings suggest a probabilistic approach to antimicrobial therapy, advocating for rapid genomic diagnostics alongside susceptibility testing to better anticipate and respond to evolutionary changes. SignificanceAntibiotic treatment can influence bacterial evolution, altering the effectiveness of subsequent therapies by inducing collateral resistance or sensitivity. This study reveals that evolution toward vancomycin-intermediate resistance in the pathogen Staphylococcus aureus proceeds through at least two distinct evolutionary pathways: one characterized by alterations in cell wall metabolism and another by changes in global stress response. These adaptive trajectories result in contrasting collateral sensitivities to first-line antibiotics. By introducing the Collateral Response Score, we assess the uncertainty in these outcomes, providing a probabilistic framework to evaluate how past antibiotic exposure shapes future treatment responses. Further validation studies are needed; however, we believe that improved forecasting of pathogen evolution can enhance antibiotic stewardship, inform therapeutic decisions, and ultimately improve patient outcomes.

evolutionary biology↗

bioRxiv2023

The Structural Role of N170 in Substrate-assisted Deacylation in KPC-2 β-Lactamase

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) results in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and altered spectrum of enzymatic activity.

biophysics↗

bioRxiv2022

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

{beta}-lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of {beta}-lactamases among pathogenic bacteria limits the efficacy of {beta}-lactam antibiotics. This has created a major public health crisis. The use of {beta}-lactamase inhibitors has proven useful in restoring the activity of {beta}-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-{beta}-lactamases (MBLs) are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the {beta}-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, 3-{beta}7 and {beta}12-5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics (MD) simulations were performed, Markov State Models (MSM) were built, and convolutional variational autoencoder (CVAE)-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between 3-{beta}7 and {beta}12-5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

biophysics↗

bioRxiv2022

The Iron Content of Human Serum Albumin Modulates the Susceptibility of Acinetobacter baumannii to Cefiderocol

Mortality rates of patients infected with Acinetobacter baumannii treated with cefiderocol (CFDC) were not as favorable as the best available treatment for pulmonary and bloodstream infections. Previous studies showed that the presence of human serum albumin (HSA) or HSA-containing fluids like human pleural fluid (HPF) or human serum (HS) in the growth medium is correlated with a decrease in the expression of genes associated with high-efficiency iron uptake systems. These observations may explain the less-than-ideal performance of CFDC in pulmonary and bloodstream infections because ferric siderophore transporters enhance penetration of CFDC into the cells cytosol. Removal of HSA from HPF or HS resulted in a reduction of the minimal inhibitory concentration of CFDC. Concomitant with these results, there was an enhancement of the expression of genes associated with high-efficiency iron uptake systems. In addition to inducing modifications in iron-uptake gene expression, removal of HSA also decreased the expression of {beta}-lactam resistance genes. Taken together, these observations indicate that environmental HSA has a role in the expression levels of selected A. baumannii. Furthermore, removal of iron from HSA had the same effect as removal of HSA on the expression of genes associated with high-efficiency iron uptake systems, suggesting that at least one of the mechanisms by which HSA regulates the expression of selected genes is through acting as an iron supplier. IMPORTANCECefiderocol (CFDC) is a new antibiotic that combines its major bactericidal activity, i.e., inhibition of the Gram-negative bacterial cell wall synthesis, with a first in its class mechanism of cell penetration. The siderophore-like moiety facilitates entry through receptors that recognize ferric-siderophore complexes. Recent trials showed that treating pulmonary and bloodstream Acinetobacter baumannii infections with CFDC did not result in the same outcomes as treating other pathogens. Our studies indicated that exposure to human fluids that contain human serum albumin (HSA) increases the MIC values of CFDC. Results described in this work show that HSA is responsible for a reduction in susceptibility of A. baumannii to CFDC. Furthermore, the presence of HSA in the milieu produces a reduction in levels of expression of proteins associated with high-affinity iron uptake systems and enhanced expression of {beta}-lactam resistance-associated genes. Deferration of HSA was accompanied by a loss of the ability to modify these genes expression levels. These results indicate that the microbiological activity of CFDC towards A. baumannii is attenuated in the presence of HSA-containing fluids. This unique insight opens up new avenues of investigation. Understanding this phenomenons molecular mechanism will help define methodologies to increase treatment efficiency.

microbiology↗

bioRxiv2021

Carbapenem-Resistant Acinetobacter baumannii in US hospitals: diversification of circulating lineages and antimicrobial resistance

Carbapenem-resistant Acinetobacter baumannii (CRAb) are a major cause of healthcare-associated infections. CRAb are typically multidrug-resistant and infection is difficult to treat. Despite the urgent threat that CRAb pose, few systematic studies of CRAb clinical and molecular epidemiology have been conducted. The Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP) is designed to investigate the clinical characteristics and contemporary population structure of CRAb circulating in US hospital systems using whole genome sequencing (WGS). Analysis of the initial 120 SNAP patients from four US centers revealed that CRAb remain a significant threat to hospitalized patients, affecting the most vulnerable patients and resulting in 24% all-cause 30-day mortality. The majority of currently circulating isolates belonged to ST2Pas, a part of Clonal Complex 2 (CC2), which is the dominant drug-resistant lineage in the United States and Europe. We identified three distinct sub-lineages within CC2, which differed in their antibiotic resistance phenotypes and geographic distribution. Most concerning, colistin resistance (38%) and cefiderocol (10%) resistance were common within CC2 sub-lineage C (CC2C), where the majority of isolates belonged to ST2Pas/ST281Ox. Additionally, we identified a newly emergent lineage, ST499Pas that was the most common non-CC2 lineage in our study and had a more favorable drug susceptibility profile compared to CC2. Our findings suggest a shift within the CRAb population in the US during the past 10 years, and emphasize the importance of real-time surveillance and molecular epidemiology in studying CRAb dissemination and clinical impact. ImportanceCarbapenem-resistant Acinetobacter baumannii (CRAb) constitute a major threat to public health. To elucidate the molecular and clinical epidemiology of CRAb in the US, clinical CRAb isolates were collected along with data on patient characteristics and outcomes and bacterial isolates underwent whole genome sequencing and antibiotic susceptibility phenotyping. Key findings included emergence of new sub-lineages within the globally predominant clonal complex (CC) 2, increased colistin and cefiderocol resistance within one of the CC2 sub-lineages, and the emergence of ST499Pas as a previously unrecognized CRAb lineage in US hospitals.

microbiology↗

bioRxiv2021

Impact of Human Serum Proteins on Susceptibility of Acinetobacter baumannii to Cefiderocol: role of iron transport

Cefiderocol is a siderophore antibiotic that co-opts iron transporters to facilitate cell entry. We show that genes related to iron uptake systems and resistance to {beta}-lactams in Acinetobacter baumannii have altered expression levels in the presence of human serum, human serum albumin, or human pleural fluid. Cefiderocol MICs are also raised in the presence of the mentioned fluids. Clinical response in A. baumannii infections may be related to the interplay of these human factors.

microbiology↗