RESUMO
This work discloses a unique, comprehensive proteomic dataset of Acinetobacter baumannii strains, both resistant and non-resistant to polymyxin B, isolated in Brazil generated using Orbitrap Fusion Lumos. From nearly 4 million tandem mass spectra, the software DiagnoMass produced 240,685 quality-filtered mass spectral clusters, of which PatternLab for proteomics identified 44,553 peptides mapping to 3479 proteins. Crucially, DiagnoMass shortlisted 3550 and 1408 unique mass spectral clusters for the resistant and non-resistant strains, respectively, with only about a third with sequences (and PTMs) identified by PatternLab. Further open-search attempts via FragPipe yielded an additional â¼20% identifications, suggesting the remaining unidentified spectra likely arise from complex combinations of post-translational modifications and amino-acid substitutions. This highlights the untapped potential of the dataset for future discoveries, particularly given the importance of PTMs, which remain elusive to nucleotide sequencing approaches but are crucial for understanding biological mechanisms. Our innovative approach extends beyond the identifications that are typically subjected to the bias of a search engine; we discern which spectral clusters are differential and subject them to increased scrutiny, akin to spectral library matching by comparing captured spectra to themselves. Our analysis reveals adaptations in the resistant strain, including enhanced detoxification, altered protein synthesis, and metabolic adjustments. SIGNIFICANCE: We present comprehensive proteomic profiles of non-resistant and resistant Acinetobacter baumannii from Brazilian Hospitals strains, and highlight the presence of discriminative and yet unidentified mass spectral clusters. Our work emphasizes the importance of exploring this overlooked data, as it could hold the key to understanding the complex dynamics of antibiotic resistance. This approach not only informs antimicrobial stewardship efforts but also paves the way for the development of innovative diagnostic tools. Thus, our findings have profound implications for the field, as far as methods for providing a new perspective on diagnosing antibiotic resistance as well as classifying proteomes in general.
Assuntos
Acinetobacter baumannii , Polimixinas , Polimixinas/metabolismo , Antibacterianos/farmacologia , Acinetobacter baumannii/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Brasil , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade MicrobianaRESUMO
The genus Acinetobacter encompasses biotechnologically relevant species and nosocomial pathogens. In this study, nine isolates recovered from different oil reservoir samples showed the ability to grow with petroleum as the only carbon source and possessed the ability to emulsify kerosene. The whole genomes of the nine strains were sequenced and analyzed. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all strains were compared to the reference strains, and the results were below the reference values (<97.88 and 82, respectively), suggesting that the isolates belong to a new subspecies of Acinetobacter baumannii. The name Acinetobacter baumannii oleum ficedula is proposed. A comparison of the whole genome repertoire of 290 Acinetobacter species indicated that the strains in this study resemble non-pathogenic Acinetobacter strains. However, the new isolates resemble A. baumannii when comparing virulence factors. The isolates in this study carry many genes involved in hydrocarbon degradation, indicating the potential to degrade most toxic compounds listed by environmental regulatory agencies such as ATSDR, EPA, and CONAMA. In addition, despite the absence of known biosurfactant or bioemulsifier genes, the strains showed emulsifying activity, suggesting the presence of new pathways or genes related to this process. This study investigated the genomic, phenotypic, and biochemical features of the novel environmental subspecies A. baumannii oleum ficedula, revealing their potential to degrade hydrocarbons and to produce biosurfactants or bioemulsifiers. Applying these environmental subspecies in bioaugmentation strategies sheds light on future approaches to bioremediation. The study shows the importance of genomic analysis of environmental strains and their inclusion in metabolic pathways databases, highlighting unique enzymes/alternative pathways for consuming hazardous hydrocarbons.
Assuntos
Acinetobacter baumannii , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Campos de Petróleo e Gás , Hidrocarbonetos/metabolismo , Genômica , DNARESUMO
We have previously shown that Acinetobacter baumannii as well as other relevant clinical bacterial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, perceive and respond to light at 37 °C, the normal temperature in mammal hosts. In this work, we present evidence indicating that the two-component system BfmRS transduces a light signal in A. baumannii at this temperature, showing selective involvement of the BfmR and BfmS components depending on the specific cellular process. In fact, both BfmR and BfmS participate in modulation of motility by light, while only BfmR is involved in light regulation of desiccation tolerance in this microorganism. Neither BfmR nor BfmS contain a photoreceptor domain and then most likely, the system is sensing light indirectly. Intriguingly, this system inhibits blsA expression at 37 °C, suggesting antagonistic functioning of both signaling systems. Furthermore, we present evidence indicating that the phosphorylatable form of BfmR represses motility. Overall, we provide experimental evidence on a new biological function of this multifaceted system that broadens our understanding of A. baumannii's physiology and responses to light.
Assuntos
Acinetobacter baumannii , Biofilmes , Animais , Humanos , Proteínas de Bactérias/metabolismo , Acinetobacter baumannii/metabolismo , Dessecação , Transdução de Sinal Luminoso , Mamíferos/metabolismoRESUMO
Infections caused by multidrug-resistant A. baumannii are a worldwide health concern with high mortality rates. Rapid identification of this infectious agent is critical as it can easily spread with difficult or no options for treatment. In this context, the development of reliable and economically viable detection and therapeutic methodologies are still challenging. One of the promising solutions is the development of nucleic acid aptamers capable of interacting with bacteria. These aptamers can be used for specific recognition of infectious agents as well as for blocking their functions. Cell-SELEX technology currently allows the selection and identification of aptamers and is flexible enough to target molecules present in an entire bacterial cell without their prior knowledge. However, the aptamer technology is still facing many challenges, such as the complexity of the screening process. Here, we describe the selection and identification of a new aptamer A01, using an in-house whole-cell SELEX-based methodology, against multi-resistant Acinetobacter baumannii, with rapid execution and low cost. In addition, this protocol allowed the identification of the aptamer A01 with the whole A. baumannii cell as a target. The aptamer A01 demonstrated a binding preference to A. baumannii when compared to K. pneumoniae, C. albicans, and S. aureus in fluorescence assays. Although the time-kill assay did not show an effect on bacterial growth, the potential bactericidal or bacteriostatic cannot be totally discarded. The new categorized aptamer (A01) displayed a significant binding affinity to MDR A. baumannii.
Assuntos
Acinetobacter baumannii , Aptâmeros de Nucleotídeos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Técnica de Seleção de Aptâmeros/métodos , Staphylococcus aureus/metabolismoRESUMO
Acinetobacter nosocomialis is a Gram-negative opportunistic pathogen, whose ability to cause disease in humans is well recognized. Blue light has been shown to modulate important physiological traits related to persistence and virulence in this microorganism. In this work, we characterized the three Blue Light sensing Using FAD (BLUF) domain-containing proteins encoded in the A. nosocomialis genome, which account for the only canonical light sensors present in this microorganism. By focusing on a light-modulated bacterial process such as motility, the temperature dependence of light regulation was studied, as well as the expression pattern and spectroscopic characteristics of the different A. nosocomialis BLUFs. Our results show that the BLUF-containing proteins AnBLUF65 and AnBLUF46 encode active photoreceptors in the light-regulatory temperature range when expressed recombinantly. In fact, AnBLUF65 is an active photoreceptor in the temperature range from 15°C to 37°C, while AnBLUF46 between 15°C to 32°C, in vitro. In vivo, only the Acinetobacter baumannii BlsA's ortholog AnBLUF65 was expressed in A. nosocomialis cells recovered from motility plates. Moreover, complementation assays showed that AnBLUF65 is able to mediate light regulation of motility in A. baumannii ΔblsA strain at 30°C, confirming its role as photoreceptor and in modulation of motility by light. Intra-protein interactions analyzed using 3D models built based on A. baumannii´s BlsA photoreceptor, show that hydrophobic/aromatic intra-protein interactions may contribute to the stability of dark/light- adapted states of the studied proteins, reinforcing the previous notion on the importance of these interactions in BLUF photoreceptors. Overall, the results presented here reveal the presence of BLUF photoreceptors in A. nosocomialis with idiosyncratic characteristics respect to the previously characterized A. baumannii's BlsA, both regarding the photoactivity temperature-dependency as well as expression patterns, contributing thus to broaden our knowledge on the BLUF family.
Assuntos
Acinetobacter baumannii , Acinetobacter , Acinetobacter/genética , Acinetobacter/metabolismo , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Temperatura , VirulênciaRESUMO
INTRODUCTION: In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. MATERIALS AND METHODS: A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. CONCLUSION: In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cefepima/farmacologia , Imipenem/farmacologia , Ionóforos de Próton/farmacologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/metabolismo , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Expressão Gênica , Genes MDR/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Quorum sensing modulates bacterial collective behaviors including biofilm formation, motility and virulence in the important human pathogen Acinetobacter baumannii. Disruption of quorum sensing has emerged as a promising strategy with important therapeutic potential. In this work, we show that light modulates the production of acyl-homoserine lactones (AHLs), which were produced in higher levels in the dark than under blue light at environmental temperatures, a response that depends on the AHL synthase, AbaI, and on the photoreceptor BlsA. BlsA interacts with the transcriptional regulator AbaR in the dark at environmental temperatures, inducing abaI expression. Under blue light, BlsA does not interact with AbaR, but induces expression of the lactonase aidA and quorum quenching, consistently with lack of motility at this condition. At temperatures found in warm-blooded hosts, the production of AHLs, quorum quenching as well as abaI and aidA expression were also modulated by light, though in this case higher levels of AHLs were detected under blue light than in the dark, in a BlsA-independent manner. Finally, AbaI reduces A. baumannii's ability to kill C. albicans only in the dark both at environmental as well as at temperatures found in warm-blooded hosts. The overall data indicate that light directly modulates quorum network in A. baumannii.
Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Percepção de Quorum/genética , Acinetobacter baumannii/metabolismo , Acil-Butirolactonas/metabolismo , Biofilmes/crescimento & desenvolvimento , Cebus/microbiologia , Humanos , Luz , Células Fotorreceptoras/metabolismo , Virulência/genéticaRESUMO
Therapeutic options are limited for patients infected with Acinetobacter baumannii due to its multidrug-resistance profile. So, the search for new antimicrobials against this gram-negative bacterial pathogen has become a worldwide priority. The present study aimed to evaluate the effects of 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2·4H2O (Cu-phendione) on 26 carbapenemase-producing A. baumannii strains. The susceptibility to carbapenems was performed by detecting the metallo-beta-lactamase (MBL) genes by PCR and by determining the MIC. Also, disk diffusion method was applied to evaluate the susceptibility to other antimicrobial classes. The test compounds were evaluated on both planktonic- and biofilm-growing bacterial cells. The results revealed that all A. baumannii strains had the intrinsic blaoxa-51 gene, and at least one of the blaoxa-23 or blaoxa-24 genes. The geometric mean MIC and minimum bactericidal concentration (MBC) values, respectively, were as follows: Cu-phendione (1.56 and 2.30 µM), Ag-phendione (2.48 and 3.63 µM), phendione (9.44 and 9.70 µM), and phen (70.46 and 184.28 µM). The test compounds (at 0.5 × MIC) affected the biofilm formation and disrupted the mature biofilm, in a typically dose-dependent manner, reducing biomass and viability parameters. Collectively, silver and copper-phendione derivatives presented potent antimicrobial action against planktonic- and biofilm-forming cells of carbapenemase-producing A. baumannii.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Fenantrolinas/farmacologia , beta-Lactamases/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/metabolismo , Antibacterianos/química , Biofilmes/crescimento & desenvolvimento , Carbapenêmicos/farmacologia , Cobre/química , Cobre/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Fenantrolinas/química , Prata/química , Prata/farmacologiaRESUMO
Introduction. The therapeutic options to treat Acinetobacter baumannii infections are very limited.Aim. Our aim was to evaluate the activity of sulbactam combined directly with avibactam or the ampicillin-sulbactam/ceftazidime-avibactam combination against extensively drug-resistant A. baumannii isolates.Methodology. Extensively drug-resistant A. baumannii isolates (n=127) collected at several South American hospitals were studied. Synergy with the sulbactam/avibactam combination was assessed in all isolates using the agar dilution method. Avibactam was used at a fixed concentration of 4 mg l-1. A disc diffusion synergy test was also performed. Synergy by a time-kill experiment was performed in a selected isolate.Results. Synergy with sulbactam/avibactam was demonstrated in 124 isolates and it showed MIC values ≤4 mg l-1. This synergy was not detected in the three New Delhi metallo-ß-lactamase-harbouring isolates. Similar results were observed with the disc diffusion synergy test of ampicillin-sulbactam/ceftazidime-avibactam. In the time-kill experiments, sulbactam/avibactam showed a rapid synergistic and bactericidal activity in ampicillin-sulbactam-resistant isolates.Conclusions. This study demonstrated that the sulbactam/avibactam combination displayed synergistic activity against A. baumannii isolates. This synergy was observed when both inhibitors were also used as part of the commercially available combinations: ampicillin-sulbactam and ceftazidime-avibactam.
Assuntos
Infecções por Acinetobacter/terapia , Compostos Azabicíclicos/metabolismo , Sulbactam/farmacologia , Infecções por Acinetobacter/metabolismo , Acinetobacter baumannii/metabolismo , Ampicilina/farmacologia , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Combinação de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Quimioterapia Combinada/métodos , Humanos , Testes de Sensibilidade Microbiana , Tienamicinas/farmacologiaRESUMO
The worldwide dissemination of metallo-ß-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we report that MBL host range is determined by the impact of MBL expression on bacterial fitness. The signal peptide sequence of MBLs dictates their adaptability to each host. In uncommon hosts, inefficient processing of MBLs leads to accumulation of toxic intermediates that compromises bacterial growth. This fitness cost explains the exclusion of VIM-2 and SPM-1 from Escherichia coli and Acinetobacter baumannii, and their confinement to Pseudomonas aeruginosa. By contrast, NDMs are expressed without any apparent fitness cost in different bacteria, and are secreted into outer membrane vesicles. We propose that the successful dissemination and adaptation of MBLs to different bacterial hosts depend on protein determinants that enable host adaptability and carbapenem resistance.