Biology subjects

Tolmasky, M.

Publications and source records attributed to Tolmasky, M..

9 recordsLinked to original sources

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↗

bioRxiv2023

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell. ImportanceAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of AAC(6')-Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubilitys impact on resistance, creating opportunities for future approaches to counter resistance.

microbiology↗

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

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↗

bioRxiv2021

Interplay between meropenem and human serum albumin on expression of carbapenem resistance genes and natural competence in Acinetobacter baumannii

Acinetobacter baumannii A118, a mostly susceptible strain, and AB5075, carbapenem-resistant, were cultured in L broth or L broth with different supplements: 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5 % HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus associated genes. The combination meropenem plus 3.5% HSA produced a synergistic augmentation in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may produce stronger cellular stress, the A. baumannii responds increasing the levels of expression of genes involved in natural competence. This process may favor the acquisition of foreign DNA and accelerate evolution. ImportanceAcinetobacter baumannii causes a variety of nosocomial- and community-infections that are usually resistant to multiple antimicrobial agents. As new strains acquire more resistance genes, these infections become harder to treat, and mortality can reach up to 39%. The high genomic plasticity exhibited by A. baumannii must be the consequence of numerous mechanisms that include acquiring foreign DNA and recombination. Here, we describe the ability of A. baumannii to induce competence genes when exposed to environments that resemble those found in the human body during untreated infection or after administration of carbapenems. In this latter scenario expression of genes related to resistance also modify their expression levels such that resistance is increased. The contributions of this article are two-pronged. First, when A. baumannii is exposed to substances present during infection, it responds, augmenting the ability to capture DNA and accelerate evolution. Second, in those conditions, the bacterium also modifies the expression of resistance genes to increase its resistance levels. In summary, recognition of substances that are naturally (HSA) or artificially (treatment with carbapenems) induces A. baumannii to defend, enhancing resistance and increasing the chances of acquiring new resistance mechanisms.

microbiology↗

bioRxiv2020

Cerebrospinal fluid (CSF) boosts metabolism and virulence expression factors in Acinetobacter baumannii

In a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an "urgent threat". Infection with this bacterium manifests as different diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, a common complication of neurosurgery caused by extensively-drug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, it is necessary to identify signals, such as exposure to cerebrospinal fluid (CSF), that trigger expression of virulence factors that are associated with the successful establishment and progress of this infection. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a low-virulence isolate showed significant differences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fimbriae, pilins, and iron chelators, and virulence as determined in various model systems. Furthermore, although CSFs presence did not enhance bacterial growth, it was associated with an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery. Experiments to identify the active CSF component pointed to human serum albumin (HSA). ImportanceAcinetobacter baumannii, notorious for its multidrug resistant phenotype, overcomes nutrient deprived and desiccated conditions through its metabolic flexibility, pathogenic and physiological adaptability. Although this pathogen is commonly associated with respiratory infections, there have been a considerable amount of cases of A. baumannii bacterial meningitis. These infections are usually post-neurological surgery complications associated with high mortality rates ranging from 40 to 70%. This work describes interactions that may occur during A. baumannii infection of human cerebrospinal fluid (CSF). A. baumanniis displays capabilities to persist and thrive in a nutrient-limited environment, which also triggers the expression of virulence factors. This work also further explores A. baumanniis utilization of an essential component within CSF to trigger enhanced expression of genes associated with its pathoadaptibility in this environment.

microbiology↗

bioRxiv2019

Restoration of Susceptibility to Amikacin by 8-Hydroxyquinoline Analogs Complexed to Zinc

Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical relevance usually harbor the 6-N-acetyltransferase type Ib [AAC(6)-Ib], an enzyme that catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic conversion to susceptibility in these bacteria. We have previously observed that Zn+2 acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6)-Ib, and in complex with ionophores it effectively reduced the levels of resistance in cellulo. We compared the activity of 8-hydroxyquinoline, three halogenated derivatives, and 5-[N-Methyl-N-Propargylaminomethyl]-8-Hydroxyquinoline in complex with Zn+2 to inhibit growth of amikacin-resistant Acinetobacter baumannii in the presence of the antibiotic. Two of the compounds, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and 5,7-diiodo-8-hydroxyquinoline, showed robust inhibition of growth of the two A. baumannii clinical isolates that produce AAC(6)-Ib. However, none of the combinations had any activity on another amikacin-resistant A. baumannii strain that possesses a different, still unknown mechanism of resistance. Time-kill assays showed that the combination of clioquinol or 5,7-diiodo-8-hydroxyquinoline with Zn+2 and amikacin was bactericidal. Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or in combination with Zn+2, and amikacin to HEK293 cells did not result in significant toxicity. These results indicate that ionophores in complex with Zn+2 could be developed into potent adjuvants to be used in combination with aminoglycosides to treat Gram-negative pathogens in which resistance is mediated by AAC(6)-Ib and most probably other related aminoglycoside modifying enzymes.

microbiology↗

bioRxiv2018

Interspecies DNA acquisition by a naturally competent Acinetobacter baumannii strain

Acinetobacter baumannii is a human pathogen that frequently acquires antibiotic resistance genes leading to the emergence of multi-drug-resistant (MDR) strains. To investigate the role of transformation in the acquisition of resistance determinants by this species, the susceptible strain A118 was exposed to genomic DNA of carbapenem-resistant Klebsiella pneumoniae (CRKp). Resistant transformants were obtained and an increase in the resistance level to all {beta}-lactam antibiotics was observed. Whole genome analysis of transformant clones demonstrated the acquisition of CRKp DNA. The most frequently acquired genes correspond to mobile elements, antibiotic resistance genes, and operons involved in metabolism. Bioinformatic analyses and in silico gene flow prediction strengthen our findings, showing that a continuing exchange of genetic material between A. baumannii and K. pneumoniae occurs when they share the same niche. Our results reinforce the idea that natural transformation may play a key role in the increasing emergence of A. baumannii MDR.\n\nIMPORTANCESince the characterization of antibiotic resistance in the late 50s, antibiotic resistance propagation was classically associated with horizontal gene transfer (HGT) mediated by plasmids bearing multiple resistance genes. Here we show that, at least in the human pathogen A. baumannii, transformation also plays a major role in the acquisition of antibiotic resistance determinants. This study unravels that at least for certain pathogens the propagation of resistance genes occurs by alternative HGT mechanisms which in the past have been unappreciated.

microbiology↗

bioRxiv2017

Identification of a Small Molecule Inhibitor of the Aminoglycoside 6’-N-Acetyltransferase Type Ib [AAC(6’)-Ib] Using Mixture-Based Combinatorial Libraries

The aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6)-Ib] is the most widely distributed enzyme among AAC(6)-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides including amikacin. This enzyme is therefore ideal to target with enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin potentiated the inhibition of growth of three resistant bacteria in culture, and improved survival when used as treatment of Galleria mellonella infected with aac(6)-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains.

microbiology↗