RESUMEN
A growing body of experimental data indicates that ceragenins (CSAs), which mimic the physicochemical properties of the host's cationic antimicrobial peptide, hold promise for the development of a new group of broad-spectrum antimicrobials. Here, using a set of in vivo experiments, we assessed the potential of ceragenins in the eradication of an important etiological agent of nosocomial infections, Acinetobacter baumannii. Assessment of the bactericidal effect of ceragenins CSA-13, CSA-44, and CSA-131 on clinical isolates of A. baumannii (n = 65) and their effectiveness against bacterial cells embedded in the biofilm matrix after biofilm growth on abiotic surfaces showed a strong bactericidal effect of the tested molecules regardless of bacterial growth pattern. AFM assessment of bacterial cell topography, bacterial cell stiffness, and adhesion showed significant membrane breakdown and rheological changes, indicating the ability of ceragenins to target surface structures of A. baumannii cells. In the cell culture of A549 lung epithelial cells, ceragenin CSA-13 had the ability to inhibit bacterial adhesion to host cells, suggesting that it interferes with the mechanism of bacterial cell invasion. These findings highlight the potential of ceragenins as therapeutic agents in the development of antimicrobial strategies against bacterial infections caused by A. baumannii.
Asunto(s)
Acinetobacter baumannii , Adhesión Bacteriana , Biopelículas , Esteroides , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Humanos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Esteroides/farmacología , Esteroides/química , Adhesión Bacteriana/efectos de los fármacos , Células A549 , Antibacterianos/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiologíaRESUMEN
Acinetobacter baumannii is one of the most important pathogens worldwide. The intrinsic and acquired resistance of A. baumannii, coupled with the slow pace of novel antimicrobial drug development, poses an unprecedented and enormous challenge to clinical anti-infective therapy of A. baumannii. Recent studies in the field of pathogenicity, antibiotic resistance, and biofilms of A. baumannii have focused on the model strains, including ATCC 17978, ATCC 19606, and AB5075. However, these model strains represent only a limited portion of the heterogeneity in A. baumannii. Furthermore, variants of these model strains have emerged that show significant diversity not only at the genotypic level but also reflected in differences at the phenotypic levels of capsule, virulence, pathogenicity, and antibiotic resistance. Research on A. baumannii, a key pathogen, would benefit from a standardized approach, which characterizes heterogeneous strains in order to facilitate rapid diagnosis, discovery of new therapeutic targets, and efficacy assessment. Our study provides and describes a standardized, genomically and phenotypically heterogeneous panel of 45 different A. baumannii strains for the research community. In addition, we performed comparative analyses of several phenotypes of this panel. We found that the sequence type 2 (ST2) group showed significantly higher rates of resistance, lower fitness cost for adaptation, and yet less biofilm formation. The Macrocolony type E (MTE, flat center and wavy edge phenotype reported in the literature) group showed a less clear correlation of resistance rates and growth rate, but was observed to produce more biofilms. Our study sheds light on the complex interplay of resistance fitness and biofilm formation within distinct strains, offering insights crucial for combating A. baumannii infection. IMPORTANCE: Acinetobacter baumannii is globally notorious, and in an effort to combat the spread of such pathogens, several emerging candidate therapies have already surfaced. However, the strains used to test these therapies vary across studies (the sources and numbers of test strains are varied and often very large, with little heterogeneity). The variation complicates the studies. Furthermore, the limited standardized resources of A. baumannii strains have greatly restricted the research on the physiology, pathogenicity, and antibiotic resistance. Therefore, it is crucial for the research community to acquire a standardized and heterogeneous panel of A. baumannii. Our study meticulously selected 45 diverse A. baumannii strains from a total of 2,197 clinical isolates collected from 64 different hospitals across 27 provinces in China, providing a scientific reference for the research community. This assistance will significantly facilitate scientific exchange in academic research.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Biopelículas , Genotipo , Fenotipo , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/crecimiento & desarrollo , Antibacterianos/farmacología , Humanos , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/tratamiento farmacológico , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Virulencia/genética , Desarrollo de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Farmacorresistencia Bacteriana/genéticaRESUMEN
We have evaluated the inhibitory effects of supernatants and lysates derived from several candidate probiotics, on the growth and biofilm formation of wound pathogens, and their ability to protect human primary epidermal keratinocytes from the toxic effects of pathogens. Supernatants (neutralized and non-neutralized) and lysates (via sonication) from Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Bifidobacterium longum, Lacticaseibacillus rhamnosus GG, and Escherichia coli Nissle 1917 were tested for their inhibitory effects against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumanni. The supernatants of L. plantarum, L. rhamnosus, B. longum, and L. rhamnosus GG reduced the growth of S. aureus, E. coli, and A. baumanni. B. longum additionally inhibited P. aeruginosa growth. However, neutralized Lactobacillus supernatants did not inhibit growth and in some cases were stimulatory. Lysates of L. plantarum and L. reuteri inhibited S. pyogenes while B. longum lysates inhibited E. coli and S. aureus growth. E. coli Nissle 1917 lysates enhanced the growth of S. pyogenes and P. aeruginosa. Biofilm formation by E. coli was reduced by lysates of L. reuteri and neutralized supernatants of all candidate probiotics. P. aeruginosa biofilm formation was reduced by E. coli Nissle supernatant but increased by L. plantarum, L. reuteri, and Bifidobacterium longum lysates. L. reuteri decreased the toxic effects of S. aureus on keratinocytes while E. coli Nissle 1917 lysates protected keratinocytes from S. pyogenes toxicity. In conclusion, lactobacilli and E. coli Nissle lysates confer inhibitory effects on pathogenic growth independently of acidification and may beneficially alter the outcome of interactions between host cell-pathogen in a species-specific manner.IMPORTANCEOne of the attributes of probiotics is their ability to inhibit pathogens. For this reason, many lactobacilli have been investigated for their effects as potential topical therapeutics against skin pathogens. However, this field is in its infancy. Even though probiotics are known to be safe when taken orally, the potential safety concerns when applied to potentially compromised skin are unknown. For this reason, we believe that extracts of probiotics will offer advantages over the use of live bacteria. In this study, we have surveyed five candidate probiotics, when used as extracts, in terms of their effects against common wound pathogens. Our data demonstrate that some probiotic extracts promote the growth of pathogens and highlight the need for careful selection of species and strains when probiotics are to be used topically.
Asunto(s)
Biopelículas , Escherichia coli , Queratinocitos , Probióticos , Pseudomonas aeruginosa , Staphylococcus aureus , Humanos , Queratinocitos/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/fisiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/fisiología , Acinetobacter baumannii/crecimiento & desarrollo , Infección de Heridas/microbiologíaRESUMEN
In this study, we evaluated the antimicrobial activity of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against seven Acinetobacter baumannii strains, the majority of which were clinically relevant carbapenem-resistant A. baumannii strains. We observed the inhibitory activity of 18 (out of 114) sponge-isolated bacterial strains against all A. baumanii strains using medium-throughput solid agar overlay assays. These inhibitory strains belonged to the genera Lactococcus, Pseudomonas, and Vagococcus. In addition, this antimicrobial activity was validated through a liquid co-cultivation challenge using an inhibitory strain of each genus and a green fluorescent protein-tagged A. baumanii strain. Fluorescence measurements indicated that the growth of A. baumanii was inhibited by the sponge isolates. In addition, the inability of A. baumanii to grow after spreading the co-cultures on solid medium allowed us to characterize the activity of the sponge isolates as bactericidal. In conclusion, this study demonstrates that marine sponges are a reservoir of bacteria that deserves to be tapped for antibiotic discovery against A. baumanii.
Asunto(s)
Acinetobacter baumannii , Antibacterianos , Carbapenémicos , Pruebas de Sensibilidad Microbiana , Poríferos , Animales , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Acinetobacter baumannii/aislamiento & purificación , Poríferos/microbiología , Antibacterianos/farmacología , Carbapenémicos/farmacología , AntibiosisRESUMEN
Carbapenem resistance of Acinetobacter baumannii poses challenges to public health. Biofilm contributes to the persistence of A. baumannii cells. This study was designed to investigate the genetic relationships among carbapenem resistance, polymyxin resistance, multidrug resistance, biofilm formation, and surface-associated motility and evaluate the antibiofilm effect of polymyxin in combination with other antibiotics. A total of 103 clinical A. baumannii strains were used to determine antibiotic susceptibility, biofilm formation capacity, and motility. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting was used to determine the genetic variation among strains. The distribution of 17 genes related to the resistance-nodulation-cell division (RND)-type efflux, autoinducer-receptor (AbaI/AbaR) quorum sensing, oxacillinases (OXA)-23, and insertion sequence of ISAba1 element was investigated. The representative strains were chosen to evaluate the gene transcription and the antibiofilm activity by polymyxin B (PB) in combination with merapenem, levofloxacin, and ceftazidime, respectively. ERIC-PCR-dependent fingerprints were found to be associated with carbapenem resistance and multidrug resistance. The presence of blaOXA-23 was found to correlate with genes involved in ISAba1 insertion, AbaI/AbaR quorum sensing, and AdeABC efflux. Carbapenem resistance was observed to be negatively correlated with biofilm formation and positively correlated with motility. PB in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with deficiency in AbaI/AbaR quorum sensing. Our results not only clarify the genetic correlation among carbapenem resistance, biofilm formation, and pathogenicity in a certain level but also provide a theoretical basis for clinical applications of polymyxin-based combination of antibiotics in antibiofilm therapy. IMPORTANCE Deeper explorations of molecular correlation among antibiotic resistance, biofilm formation, and pathogenicity could provide novel insights that would facilitate the development of therapeutics and prevention against A. baumannii biofilm-related infections. The major finding that polymyxin B in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with genetic deficiency in AbaI/AbaR quorum sensing further provides a theoretical basis for clinical applications of antibiotics in combination with quorum quenching in antibiofilm therapy.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Ceftazidima/uso terapéutico , Polimixina B/uso terapéutico , Percepción de Quorum/genética , Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/crecimiento & desarrollo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Biopelículas/crecimiento & desarrollo , Ceftazidima/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Quimioterapia Combinada/métodos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Polimixina B/farmacología , Percepción de Quorum/efectos de los fármacos , beta-Lactamasas/genéticaRESUMEN
Acinetobacter baumannii has emerged globally as a difficult-to-treat nosocomial pathogen and become resistant to carbapenems, resulting in limited treatment options. KBP-7072 is a novel semisynthetic aminomethylcycline, expanded spectrum tetracycline antibacterial agent with completed phase 1 clinical development studies. This study aimed to evaluate the in vitro activity of KBP-7072 and several comparators against clinical A. baumannii isolates collected from China. A collection of 536 A. baumannii clinical isolates were isolated from 20 hospitals across 13 provinces and cities in China between 2018 and 2019. Antimicrobial susceptibility testing of 12 antimicrobial agents was performed utilizing the broth microdilution method recommended by CLSI. KBP-7072 has shown active antibacterial activity against 536 A. baumannii isolates. It inhibited the growth of all isolates at 4 mg/liter, including 372 carbapenem-resistant isolates, 37 tigecycline MIC ≥ 4 mg/liter isolates, and 138 omadacycline MIC ≥ 4 mg/liter isolates. Compared with other expanded spectrum tetracyclines, KBP-7072 (MIC90, 1 mg/liter) outperformed 2-fold and 4-fold more active against 536 A. baumannii isolates than tigecycline (MIC90, 2 mg/liter) and omadacycline (MIC90, 4 mg/liter). KBP-7072 was as equally active as colistin (MIC90, 1 mg/liter, 99.4% susceptible). Doxycycline (33.4% susceptible), gentamicin (31.3% susceptible), meropenem (30.6%, susceptible), imipenem (30.2% susceptible), ceftazidime (27.8% susceptible), piperacillin-tazobactam (27.2% susceptible), and levofloxacin (27.2% susceptible) showed marginally poor antibacterial activity against tested isolates according to CLSI breakpoints, except for minocycline (73.7% susceptible). KBP-7072 is a potential alternative agent for the treatment of infection caused by A. baumannii, including carbapenem-resistant species. IMPORTANCE It is reported that A. baumannii has emerged as an intractable nosocomial pathogen in hospitals especially when it develops resistance to carbapenems and other antibiotics, which limits treatment options and leads to high mortality. In February 2017, the WHO published a list of ESKAPE pathogens designated "priority status" for which new antibiotics are urgently needed. Therefore, the epidemiological surveillance and new therapeutic development of A. baumannii must be strengthened to confront an emerging global epidemic. KBP-7072 is a novel, expanded spectrum tetracycline antibacterial and has demonstrated good in vitro activity against recent geographically diverse A. baumannii isolates collected from North America, Europe, Latin America, and Asia-Pacific. This study has shown excellent in vitro activity of KBP-7072 against clinical A. baumannii isolates collected from different regions of China, regarded as supplementary to KBP-7072 pharmacodynamics data, which is of great significance, as it is promising an alternative treatment in CRAB isolates infections in China.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Tetraciclinas/farmacología , Acinetobacter baumannii/crecimiento & desarrollo , Carbapenémicos/farmacología , China , Farmacorresistencia Bacteriana Múltiple , Humanos , Pruebas de Sensibilidad Microbiana , Tigeciclina/farmacologíaRESUMEN
Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res 68:5318-5325, 2008) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.
Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Acinetobacter baumannii/efectos de los fármacos , Amicacina/farmacología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Compuestos Organometálicos/farmacología , Piridinas/farmacología , Acetiltransferasas/metabolismo , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/crecimiento & desarrollo , Amicacina/metabolismo , Antibacterianos/metabolismo , Inhibidores Enzimáticos/química , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/crecimiento & desarrollo , Viabilidad Microbiana , Compuestos Organometálicos/química , Piridinas/química , Solubilidad , Factores de TiempoRESUMEN
The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.
Asunto(s)
Acinetobacter baumannii/enzimología , Acinetobacter baumannii/crecimiento & desarrollo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Carboxipeptidasas/metabolismo , Pared Celular/enzimología , beta-Lactamas/farmacología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Carboxipeptidasas/genética , Pared Celular/efectos de los fármacos , Pared Celular/genética , Farmacorresistencia BacterianaRESUMEN
Nutrient acquisition systems are often crucial for pathogen growth and survival during infection, and represent attractive therapeutic targets. Here, we study the protein machinery required for heme uptake in the opportunistic pathogen Acinetobacter baumannii. We show that the hemO locus, which includes a gene encoding the heme-degrading enzyme, is required for high-affinity heme acquisition from hemoglobin and serum albumin. The hemO locus includes a gene coding for a heme scavenger (HphA), which is secreted by a Slam protein. Furthermore, heme uptake is dependent on a TonB-dependent receptor (HphR), which is important for survival and/or dissemination into the vasculature in a mouse model of pulmonary infection. Our results indicate that A. baumannii uses a two-component receptor system for the acquisition of heme from host heme reservoirs.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/metabolismo , Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/crecimiento & desarrollo , Animales , Proteínas Bacterianas/genética , Transporte Biológico , Femenino , Humanos , Ratones Endogámicos BALB C , Familia de MultigenesRESUMEN
Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.
Asunto(s)
Acinetobacter baumannii/crecimiento & desarrollo , Acinetobacter baumannii/metabolismo , Membrana Externa Bacteriana/metabolismo , Lipopolisacáridos/biosíntesis , Peptidoglicano/biosíntesis , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Membrana Externa Bacteriana/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lipopolisacáridos/química , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/química , Periplasma/química , Periplasma/genética , Periplasma/metabolismoRESUMEN
The bacterial pathogen Acinetobacter baumannii has emerged as an urgent threat to health care systems. The prevalence of multidrug resistance in this critical human pathogen is closely associated with difficulties in its eradication from the hospital environment and its recalcitrance to treatment during infection. The development of resistance in A. baumannii is in part due to substantial plasticity of its genome, facilitating spontaneous genomic evolution. Many studies have investigated selective pressures imposed by antibiotics on genomic evolution, but the influence of high-abundance bioactive molecules at the host-pathogen interface on mutation and rates of evolution is poorly understood. Here, we studied the roles of host fatty acids in the gain in resistance to common antibiotics. We defined the impact of the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid on the development of resistance to erythromycin in A. baumannii strain AB5075_UW using a microevolutionary approach. We employed whole-genome sequencing and various phenotypic analyses to characterize microbe-lipid-antibiotic interactions. Cells exposed to erythromycin in the presence of the fatty acids displayed significantly lower rates of development of resistance to erythromycin and, importantly, tetracycline. Subsequent analyses defined diverse means by which host fatty acids influence the mutation rates. This work has highlighted the critical need to consider the roles of host fatty acids in A. baumannii physiology and antimicrobial resistance. Collectively, we have identified a novel means to curb the development of resistance in this critical human pathogen. IMPORTANCE The global distribution of multidrug resistance in A. baumannii has necessitated seeking not only alternative therapeutic approaches but also the means to limit the development of resistance in clinical settings. Highly abundant host bioactive compounds, such as polyunsaturated fatty acids, are readily acquired by A. baumannii during infection and have been illustrated to impact the bacterium's membrane composition and antibiotic resistance. In this work, we show that in vitro supplementation with host polyunsaturated fatty acids reduces the rate at which A. baumannii gains resistance to erythromycin and tetracycline. Furthermore, we discover that the impact on resistance development is closely associated with the primary antimicrobial efflux systems of A. baumannii, which represent one of the major drivers of clinical resistance. Overall, this study emphasizes the potential of host macromolecules in novel approaches to circumvent the difficulties of multidrug resistance during A. baumannii treatment, with fatty acid supplements such as fish oil providing safe and cost-effective ways to enhance host tolerance to bacterial infections.
Asunto(s)
Acinetobacter baumannii/genética , Antibacterianos/farmacología , Ácido Araquidónico/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Eritromicina/farmacología , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Membrana Celular/química , Genoma Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana , Tasa de Mutación , Selección Genética/genética , Tetraciclinas/farmacología , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: The recent rise and spread of carbapenem-resistant pathogens pose an urgent threat to public health and has fueled the search for new therapies. Localized delivery of topical antibiotics is an alternative for the treatment of infected wounds caused by drug-resistant pathogens. In this study, we aimed to develop antimicrobial-loaded hydrogels for topical treatment of wound infections in a murine skin wound infection. RESULTS: Paenipeptin analogue 1, a linear lipopeptide, potentiated clarithromycin against multidrug-resistant Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae. Enzymatically-crosslinked gelatin hydrogels were developed to encapsulate paenipeptin analogue 1 and clarithromycin. The encapsulated antimicrobials were gradually released from hydrogels during incubation, reaching 75.43 and 53.66% for paenipeptin and clarithromycin, respectively, at 24 h. The antimicrobial-loaded hydrogels containing paenipeptin and clarithromycin synergistically resulted in 5-log reduction in carbapenem-resistant A. baumannii within 6 h in vitro. Moreover, the antimicrobial-loaded hydrogels reduced 3.6- and 2.5-log of carbapenem-resistant A. baumannii when treated at 4 or 20 h post infection, respectively, in a murine skin wound infection. CONCLUSIONS: Enzymatically-crosslinked gelatin hydrogels loaded with paenipeptin analogue 1 and clarithromycin exhibited potent therapeutic efficacy against carbapenem-resistant A. baumannii in murine skin wound infection.
Asunto(s)
Antibacterianos/farmacología , Claritromicina/química , Claritromicina/farmacología , Hidrogeles/química , Lipopéptidos/química , Lipopéptidos/farmacología , Infección de Heridas/tratamiento farmacológico , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Animales , Antibacterianos/química , Biocatálisis , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Femenino , Gelatina/química , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Piel/microbiología , Transglutaminasas/química , Infección de Heridas/microbiologíaRESUMEN
CsrA is a global regulator widespread in bacteria and known to be involved in different physiological processes, including pathogenicity. Deletion of csrA of Acinetobacter baumannii strain ATCC 19606 resulted in a mutant that was unable to utilize a broad range of carbon and energy sources, including amino acids. This defect in amino acid metabolism was most likely responsible for the growth inhibition of the ΔcsrA mutant in human urine, where amino acids are the most abundant carbon source for A. baumannii. Recent studies revealed that deletion of csrA in the A. baumannii strains AB09-003 and ATCC 17961 resulted in an increase in hyperosmotic stress resistance. However, the molecular basis for this observation remained unknown. This study aimed to investigate the role of CsrA in compatible solute synthesis. We observed striking differences in the ability of different A. baumannii strains to cope with hyperosmotic stress. Strains AB09-003 and ATCC 17961 were strongly impaired in hyperosmotic stress resistance in comparison to strain ATCC 19606. These differences were abolished by deletion of csrA and are in line with the ability to synthesize compatible solutes. In the salt-sensitive strains AB09-003 and ATCC 17961, compatible solute synthesis was repressed by CsrA. This impairment is mediated via CsrA and could be overcome by deletion of csrA from the genome. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and ATCC 17961. Here, we report that this is also the case for A. baumannii ATCC 19606. However, we observed significant differences in the ΔcsrA mutants with respect to osmostress resistance, such as the AB09-003 and 17961 mutants being enhanced in osmostress resistance whereas the ATCC 19606 mutant was not. This suggests that the role of CsrA in osmotic stress adaptation is strain specific. Furthermore, we provide clear evidence that CsrA is essential for growth in human urine and at high temperatures.
Asunto(s)
Acinetobacter baumannii/crecimiento & desarrollo , Adaptación Fisiológica/genética , Proteínas Bacterianas/genética , Presión Osmótica/fisiología , Orina/microbiología , Acinetobacter baumannii/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Humanos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We evaluated phytochemical composition, antibacterial, antifungal, anti-oxidant and cytotoxic properties of aqueous (water) and organic extracts (methanol, ethyl acetate and n-hexane) of Chenopodium glaucum. Highest phenolic content 45 mg gallic acid equivalents (GAE)/g d.w was found in aqueous extract followed by ethyl acetate (41mg GAE/g d.w) and methanol extract (34.46 mg GAE/g d.w). Antibacterial potential of aqueous and organic extracts of C. glaucum was examined against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Staphylococcus epidermidis. The aqueous, methanolic, ethyl acetate, and n-hexane extract showed antibacterial activity against A. baumannii, K. pneumoniae, E. coli and S. epidermidis. However, against A. baumannii significantly higher inhibition zone (19 mm and 18.96 mm respectively) was shown by ethyl acetate and methanol extracts. Aqueous extract possessed highest growth inhibition (11 mm) against E. coli. Aqueous, ethyl acetate and methanol extracts showed 9 mm, 10 mm, and 10.33 mm zone of inhibition against the K. pneumoniae. For antifungal activity, the extracts were less effective against Aspergillus niger but showed strong antifungal activity against Aspergillus flavus (A. flavus). The antioxidant activity was measured as DPPH (2, 2-diphenyl-1-picrylhydrazyl), H2O2 and ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity of free radicals. All the organic extracts of C. glaucum possessed ABTS, DPPH and H2O2 scavenging properties. The highest cytotoxic activity measured as half maximal inhibitory concentration (IC50) against human lungs carcinoma cells was recorded for methanolic (IC50 = 16 µg/mL) and n-hexane (IC50 = 25 µg/mL) extracts, respectively. The Gas chromatography-mass spectrometry (GC-MS) analysis showed 4 major and 26 minor compounds in n-hexane extract and 4 major and 7 minor compounds in methanol extract of the C. glaucum. It is concluded that aqueous and organic extracts of C. glaucum would be potential therapeutic agents and could be exploited on a pilot scale to treat human pathogenic diseases.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antineoplásicos/farmacología , Chenopodium/química , Neoplasias Pulmonares/tratamiento farmacológico , Extractos Vegetales/farmacología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Antioxidantes/farmacología , Aspergillus/efectos de los fármacos , Aspergillus/crecimiento & desarrollo , Línea Celular Tumoral , Citotoxinas/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Fitoquímicos/farmacología , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/crecimiento & desarrolloRESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAB) has been a common pathogen of nosocomial infections and severely threatened the public health for decades. Tigecycline is a new type of antibacterial glycylcycline and minocycline derivative and has been used to treat CRAB in clinical practice. However, the synergistic effects of tigecycline in combination with other antibiotics including colistin or amikacin remain unclear. A total of 216 CRAB isolates were collected from multiple body parts of different patients. The gene types of these isolates were analyzed and their resistance to carbapenems was determined by Etest. Broth microdilution method was utilized to evaluate the minimum inhibitory concentration (MIC) of each sample. Checkerboard screening technique was performed to demonstrate the synergistic effects of antibiotics and fractional inhibitory concentration index (FICI) was established. Therefore, the joint treatment of tigecycline and colistin (1:1) could effectively improve the sensitivity of AB to antibiotics. OXA-24-like isolates were more sensitive to the combination of tigecycline and amikacin. On the other hand, OXA-23-like isolates were more sensitive to the combination of tigecycline and colistin. Tigecycline exhibited synergistic effects with amikacin and colistin to inhibit CRAB.
Asunto(s)
Acinetobacter baumannii/crecimiento & desarrollo , Amicacina/farmacología , Carbapenémicos , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Tigeciclina/farmacología , Resistencia betalactámica/efectos de los fármacos , Amicacina/agonistas , Colistina/agonistas , Sinergismo Farmacológico , Tigeciclina/agonistasRESUMEN
Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol, and bronopol) were classified via checkerboard assay and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443, and Staphylococcus aureus NCTC 13143. Synergistic interactions were identified only for the combinations of chlorocresol with benzalkonium chloride and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterized as additive. We suggest that this is due to the broad, nonspecific mechanisms associated with disinfectants not providing an opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCE The scarcity of observed synergistic interactions suggests that in the case of many disinfectant-based products, combined mechanisms of interaction may be being misinterpreted. We emphasize the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.
Asunto(s)
Compuestos de Benzalconio/farmacología , Biguanidas/farmacología , Cresoles/farmacología , Desinfectantes/farmacología , Glicoles de Propileno/farmacología , Compuestos de Amonio Cuaternario/farmacocinética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Sinergismo Farmacológico , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/crecimiento & desarrollo , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Compuestos de Amonio Cuaternario/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
History, chance, and selection are the fundamental factors that drive and constrain evolution. We designed evolution experiments to disentangle and quantify effects of these forces on the evolution of antibiotic resistance. Previously, we showed that selection of the pathogen Acinetobacter baumannii in both structured and unstructured environments containing the antibiotic ciprofloxacin produced distinct genotypes and phenotypes, with lower resistance in biofilms as well as collateral sensitivity to ß-lactam drugs (Santos-Lopez et al., 2019). Here we study how this prior history influences subsequent evolution in new ß-lactam antibiotics. Selection was imposed by increasing concentrations of ceftazidime and imipenem and chance differences arose as random mutations among replicate populations. The effects of history were reduced by increasingly strong selection in new drugs, but not erased, at times revealing important contingencies. A history of selection in structured environments constrained resistance to new drugs and led to frequent loss of resistance to the initial drug by genetic reversions and not compensatory mutations. This research demonstrates that despite strong selective pressures of antibiotics leading to genetic parallelism, history can etch potential vulnerabilities to orthogonal drugs.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Evolución Biológica , Farmacorresistencia Bacteriana , Acinetobacter baumannii/genética , Acinetobacter baumannii/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Ciprofloxacina/farmacología , Exposición a Riesgos Ambientales , Humanos , Mutación , Selección GenéticaRESUMEN
The host defense peptide caerin 1.9 was originally isolated from skin secretions of an Australian tree frog and inhibits the growth of a wide range of bacteria in vitro. In this study, we demonstrated that caerin 1.9 shows high bioactivity against several bacteria strains, such as Staphylococcus aureus, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus haemolyticus in vitro. Importantly, unlike the antibiotic Tazocin, caerin 1.9 does not induce bacterial resistance after 30 rounds of in vitro culture. Moreover, caerin 1.1, another peptide of the caerin family, has an additive antibacterial effect when used together with caerin 1.9. Furthermore, caerin 1.1 and 1.9 prepared in the form of a temperature-sensitive gel inhibit MRSA growth in a skin bacterial infection model of two murine strains. These results indicate that caerin 1.1 and 1.9 peptides could be considered an alternative for conventional antibiotics. IMPORTANCE Antibiotic-resistant bacteria cause severe problems in the clinic. We show in our paper that two short peptides isolated from an Australian frog and prepared in the form of a gel are able to inhibit the growth of antibiotic-resistant bacteria in mice, and, unlike antibiotics, these peptides do not lead to the development of peptide-resistant bacteria strains.
Asunto(s)
Proteínas Anfibias/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Infecciones Bacterianas/microbiología , Farmacorresistencia Bacteriana , Piel/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Animales , Anuros , Australia , Infecciones Bacterianas/tratamiento farmacológico , Femenino , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Piel/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
BACKGROUND: Melittin is one of the most studied antimicrobial peptides, and several in vitro experiments have demonstrated its antibacterial efficacy. However, there is evidence showing melittin has non-promising effects such as cytotoxicity and hemolysis. Therefore, concerns about unwanted collateral toxicity of melittin lie ahead in the path toward its clinical development. With these considerations, the present study aimed to fill the gap between in vitro and in vivo studies. METHODS: In the first step, in vitro toxicity profile of melittin was assessed using cytotoxicity and hemolysis tests. Next, a maximum intraperitoneal (i.p.) sub-lethal dose was determined using BALB/c mice. Besides toxicity, antimicrobial efficacy of melittin against extensively drug-resistant (XDR) Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and KPC-producing Klebsiella pneumonia (KPC-KP) pathogens were tested using both in vitro and in vivo methods. RESULTS: Melittin showed extensive hemolysis (HD50 = 0.44 µg/mL), and cytotoxicity (IC50 = 6.45 µg/mL) activities with i.p. LD50 value of 4.98 mg/kg in BALB/c mice. In vitro antimicrobial evaluation showed melittin MIC range from 8 to 32 µg/mL for the studied pathogens. Treatment of infected mice with repeated sub-lethal doses of melittin (2.4 mg/kg) displayed no beneficial effect on their survival and peritoneal bacterial loads. CONCLUSIONS: These results indicate that melittin at its safe dose could not exhibit antimicrobial activity, which hinders its application in clinical practice.
Asunto(s)
Antibacterianos/toxicidad , Meliteno/toxicidad , Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/crecimiento & desarrollo , Animales , Antibacterianos/uso terapéutico , Línea Celular , Farmacorresistencia Bacteriana , Hemólisis/efectos de los fármacos , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Masculino , Meliteno/uso terapéutico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Peritonitis/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
The emergence of carbapenem-resistant Acinetobacter baumannii has increased the risk of nosocomial infections, which pose a huge health threat. There is an urgent need to develop alternative therapies, including broad-spectrum antimicrobial peptides. In this study, we designed, characterized, and studied the antibacterial, antibiofilm effects and possible mode of actions of a novel synthetic peptide Octopromycin, derived from the proline-rich protein 5 of Octopus minor. Octopromycin consists of 38 amino acids, (+5) net positive charge, high hydrophobic residue ratio (36%), and two α-helix secondary structures. The minimum inhibitory concentration and minimum bactericidal concentration against A. baumannii were 50 and 200 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octopromycin. Field emission scanning electron microscopy images clearly showed ultrastructural alterations in Octopromycin-treated A. baumannii cells. Propidium iodide penetrated into Octopromycin-treated A. baumannii cells, demonstrating the loss of cell membrane integrity. Octopromycin treatment increased the production of reactive oxygen species in a concentration-dependent manner, and it inhibited the biofilm formation and showed biofilm eradication activity against A. baumannii. In vitro and in vivo safety evaluation revealed that Octopromycin was nontoxic to HEK293T and Raw 264.7 cells (<400 µg/mL), as well as mice red blood cells (<300 µg/mL), and zebrafish embryos (<4 µg/mL). An in vivo study results revealed that the A. baumannii-infected fish treated with Octopromycin exhibited a significantly higher relative percent survival (37.5%) than the infected mock-treated fish with PBS (16.6%). Furthermore, a decreased bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octopromycin in vivo. Collectively, the results indicate that the antibacterial peptide Octopromycin may achieve rapid control of A. baumannii through multi-target interactions; it presents a desirable therapeutic option for the prevention and control of the infections.