Biology subjects

Bonomo, R. A.

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

28 records · Page 2Linked to original sources

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↗

bioRxiv2021

β-lactamase remodeling and evolution of collateral resistance in hypermutator Pseudomonas aeruginosa upon long-term antibiotic therapy

Antibiotic resistance development has been studied using approaches that range from laboratory experimental evolution, surveillance and epidemiology, to clinical isolate sequencing. However, evolutionary trajectories depend on the environment in which selection takes place, compelling to address evolutionary analyses in antibiotic-treated patients, to embrace the whole inherent environmental complexities as well as their dynamics over time. Herein, we address the complexity of the bacterial adaptive response to changing antibiotic selective pressures by studying the long-term in-patient evolution of a broad diversity of {beta}-lactam resistant Pseudomonas aeruginosa clones. By using mutational and ultra-deep amplicon sequencing, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis (CF) patient. We identified the accumulation of multiple alterations targeting the chromosomally encoded class C {beta}-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, "a gain of function" of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the diversity of evolutionary trajectories driven by bacteria in the natural CF environmental setting, towards a multidrug resistant phenotype after years of antibiotic treatment against a formidable pathogen.

microbiology↗

bioRxiv2021

Specific protein-membrane interactions promote the export of metallo-β-lactamases via outer membrane vesicles

Outer membrane vesicles (OMVs) act as carriers of resistance determinants such as metallo-{beta}-lactamases. The metallo-{beta}-lactamase NDM-1 is present in OMVs produced by Gram-negative bacteria since it is a lipidated, membrane-anchored protein. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Herein, we show that these interactions promote its export into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1, while being absent in VIM-2. These interactions correlate with an enhanced secretion into OMVs of IMP-1 compared to VIM-2. Disruption of these interactions in NDM-1 and IMP-1 impairs export into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that export of metallo-{beta}-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on molecular features.

molecular biology↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗

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↗

bioRxiv2020

The H-NS regulator plays a role in the stress induced by carbapenemase expression in Acinetobacter baumannii

Disruption of the histone-like nucleoid structuring protein (H-NS) was shown to affect the ability for Gram-negative bacteria to regulate genes associated with virulence, persistence, stress response, quorum sensing, biosynthesis pathways and cell adhesion. Here, we used the expression of metallo-{beta}-lactamases (MBLs) known to elicit envelope stress by the accumulation of toxic species in the periplasm to interrogate the role of H-NS in Acinetobacter baumannii, together with other stressors. Using a multidrug-resistant A. baumannii, we observed that H-NS plays a role in alleviating the stress triggered by MBL toxic precursors and counteract the effect of DNA-damaging agents, supporting its role in stress response. ImportanceCarbapenem-resistant A. baumannii (CRAB) is recognized as one of the most threatening gram-negative bacilli. H-NS is known to play a role in controlling the transcription of a variety of different genes, including those associated with stress response, persistence and virulence. In the present work, we uncovered a link between the role of H-NS in the A. baumannii stress response and its relationship with the envelope stress response and resistance to DNA-damaging agents. Overall, we posit a new role of H-NS, showing that H-NS serves to endure envelope stress that could also be a mechanism that alleviates the stress induced by MBL expression in A. baumannii. This could be an evolutionary advantage to further resist the action of carbapenems.

microbiology↗

bioRxiv2018

The Clinical and Molecular Epidemiology of CTX-M-9 Group Producing Enterobacteriaceae infections in children

BackgroundThe pandemic of extended-spectrum-beta-lactamase (ESBL)-producing-Enterobacteriaceae (Ent) is strongly linked to the dissemination of CTX-M-type-ESBL-Ent. We sought to define the epidemiology of infections in children due to an emerging resistance type, CTX-M-9-group-producing-Ent (CTX-M-9-grp-Ent).\n\nMethodsA multi-centered case-control analysis of Chicago children with CTX-M-9-grp-Ent infections was performed. Cases were defined as children possessing extended-spectrum-cephalosporin-resistant (ESC-R) infections. PCR and DNA analysis assessed beta-lactamase (bla) genes, multi-locus sequence types (MLST) and phylogenetic grouping of E. coli. Controls were children with ESC-susceptible (ESC-S)-Ent infections matched 3:1 by age, source, and hospital. The clinical-epidemiologic predictors of CTX-M-9-grp-Ent infection were assessed.\n\nResultsOf 356 ESC-R-Ent isolates from children (median age 4.1 years), CTX-M-9-group was the solely detected bla gene in 44(12.4%). The predominant species was E. coli (91%) of virulent phylogroups D(60%) and B2(40%). MLST revealed multiple strain types. On multivariable analysis, CTX-M-9-grp-Ent occurred more often in E. coli (OR 7.0), children of non-black-white-Hispanic race (OR 6.5), and outpatients (OR 4.5) which was a very unexpected finding for infections due to antibiotic-resistant bacteria. Residents of South Chicago were 6.7 times more likely to have CTX-M-9-grp-Ent infections than those in the reference region (West), while residence in Northwestern Chicago was associated with an 81% decreased risk. Other demographic, comorbidity, invasive-device, and antibiotic use differences were not found.\n\nConclusionsCTX-M-9-grp-Ent infection is strikingly associated with patient residence and is occurring in children without traditional in-patient exposure risk factors. This suggests that among children, the community environment may be a key contributor in the spread of these resistant pathogens.

epidemiology↗

bioRxiv2018

A non-canonical metal center drives activity of the Sediminispirochaeta smaragdinae metallo-β- lactamase SPS-1

In an effort to evaluate whether a recently reported putative metallo-{beta}-lactamase (M{beta}L) contains a novel M{beta}L active site, SPS-1 from Sediminispirochaeta smaragdinae was over-expressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equivalents of Zn (II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not {beta}-lactam substrates with bulky substituents in the 6-7 position. Spectroscopic studies on Co (II)-substituted SPS-1 suggest a novel metal center in SPS-1, with reduced spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar that that in NDM-1 and other subclass B1 M{beta}Ls; however, the Zn1 metal ion is coordinated by 2 histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 [A] from the position reported in other M{beta}Ls. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents in the 6/7 position of {beta}-lactam antibiotics. This study reveals a novel metal binding site in M{beta}Ls, and suggests that the targeting of metal binding sites in M{beta}Ls with inhibitors is now more challenging with the identification of this new M{beta}L.

biochemistry↗

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↗

bioRxiv2018

Community origins and regional differences in plasmid-mediated fluoroquinolone resistant Enterobacteriaceae infections in children

BackgroundFluoroquinolones (FQs) are uncommonly prescribed in children, yet pediatric multidrug-resistant (MDR)-Enterobacteriaceae (Ent) infections often reveal FQ resistance (FQR). We sought to define the molecular epidemiology of FQR and MDR-Ent in children.\n\nMethodsA case-control analysis of children with MDR-Ent infections at 3 Chicago hospitals was performed. Cases were children with third-generation-cephalosporin-resistant (3GCR) and/or carbapenem-resistant (CR)-Ent infections. PCR and DNA analysis assessed bla and plasmid-mediated FQR (PMFQR) genes. Controls were children with 3GC and carbapenem susceptible-Ent infections matched by age, source and hospital. We assessed clinical-epidemiologic predictors of PMFQR Ent infection.\n\nResultsOf 169 3GCR and/or CR Ent isolates from children (median age 4.8 years), 85 were FQR; 56 (66%) contained PMFQR genes. The predominant organism was E. coli and most common bla gene bla CTX-M-1 group. In FQR isolates, PMFQR gene mutations included aac61b-cr, oqxA/B, qepA, and qnrA/B/D/S in 83%, 15%, 13% and 11% of isolates, respectively. FQR E. coli was often associated with phylogroup B2, ST43/ST131. On multivariable analysis, PMFQR Ent infections occurred mostly in outpatients (OR 33.1) of non-black-white-Hispanic race (OR 6.5). Residents of Southwest Chicago were >5 times more likely to have PMFQR-Ent infections than those in the reference region, while residence in Central Chicago was associated with a 97% decreased risk. Other demographic, comorbidity, invasive-device, antibiotic use, or healthcare differences were not found.\n\nConclusionsThe strong association of infection with MDROs showing FQR with patient residence rather than with traditional risk factors suggests that the community environment is a major contributor to spread of these pathogens in children.

epidemiology↗