RESUMO
Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.
Assuntos
Antibacterianos , Proteínas de Bactérias , Klebsiella pneumoniae , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Peptidoglicano , Plasmídeos , beta-Lactamases , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Klebsiella pneumoniae/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Plasmídeos/genética , Animais , Infecções por Klebsiella/microbiologia , Mariposas/microbiologiaRESUMO
Inhaled corticosteroids (ICS) use is associated with an increased risk of Pseudomonas aeruginosa (PA) infection in patients with COPD. We aimed to evaluate the effects of ICS on alveolar macrophages in response to PA in COPD patients with and without baseline ICS treatment (COPD and COPD + ICS, respectively) as well as smoker and nonsmoker controls. To do so, cells were infected with PA and cotreated with budesonide (BUD) or fluticasone propionate (FLU). The analysis of NF-κB and c-jun activity revealed a significant increase in both factors in response to PA cotreated with BUD/FLU in smokers but not in COPD or COPD + ICS patients when compared with PA infection alone. The expression of Toll-like receptor 2 (TLR2) and the transcription factor c-jun were induced upon PA infection in nonsmokers only. Moreover, in the smoker and COPD groups, there was a significant increase in TLR2 and a decrease in c-jun expression when treated with BUD/FLU after PA infection, which were not observed in COPD + ICS patients. Therefore, the chronic use of ICS seemingly makes the macrophages tolerant to BUD/FLU stimulation compared with those from patients not treated with ICS, promoting an impaired recognition of PA and activity of alveolar macrophages in terms of altered expression of TLR2 and cytokine production, which could explain the increased risk of PA infection in COPD patients under ICS treatment.
Assuntos
Infecções por Pseudomonas , Doença Pulmonar Obstrutiva Crônica , Administração por Inalação , Corticosteroides/efeitos adversos , Budesonida/efeitos adversos , Humanos , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptor 2 Toll-LikeRESUMO
BACKGROUND: Mycoplasma pneumoniae (MP) causes community-acquired pneumonia affecting mainly children, and tends to produce cyclic outbreaks. The widespread use of macrolides is increasing resistance rates to these antibiotics. Molecular tools can help in diagnosis, typing and resistance detection, leading to better patient management. OBJECTIVES: To assess the MP genotypes and resistance pattern circulating in our area while comparing serological and molecular diagnosis of MP. METHODS: Molecular and serological diagnosis of MP was performed in 821 samples collected in Badalona (Barcelona, Spain) from 2013 to 2017. Multiple locus variable number tandem repeat analysis (MLVA) and macrolide resistance detection by pyrosequencing were performed in those cases positive by PCR. Presence of respiratory viruses and relevant clinical data were also recorded. RESULTS: MP was detected in 16.8% of cases by PCR, with an overall agreement with serology of 76%. Eleven different MLVA types were identified, with 4-5-7-2 (50.1%) and 3-5-6-2 (29.2%) being the most abundant, with the latter showing a seasonal increase during the study. A total of 8% of the strains harboured a point substitution associated with macrolide resistance, corresponding mainly to an A2063G 23S rRNA mutation and directly related to previous macrolide therapy. Analysis of respiratory viruses showed viral coinfections in most cases. CONCLUSIONS: Serological and molecular tools combined could improve MP diagnosis and the analysis of its infection patterns. Macrolide resistance is associated with previous therapy. Given that MP pneumonia usually resolves spontaneously, it should be reconsidered whether antibiotic treatment is suitable for all cases.
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Antibacterianos , Pneumonia por Mycoplasma , Adolescente , Adulto , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Humanos , Macrolídeos/farmacologia , Masculino , Tipagem Molecular , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/epidemiologia , RNA Ribossômico 23S/genética , Espanha/epidemiologiaRESUMO
BACKGROUND: Searching for new strategies to defeat Pseudomonas aeruginosa is of paramount importance. Previous works in vitro showed that peptidoglycan recycling blockade disables AmpC-dependent resistance and enhances susceptibility against cell-wall-targeting immunity. Our objective was to validate these findings in murine models.This study shows for the first time in different murine models of infection that blocking the peptidoglycan recycling in Pseudomonas aeruginosa causes an important virulence impairment and disables AmpC-mediated resistance, being hence validated as a promising therapeutic target. METHODS: Wildtype PAO1, recycling-defective AmpG and NagZ mutants, an AmpC hyperproducer dacB mutant, and their combinations were used to cause systemic/respiratory infections in mice. Their survival, bacterial burden, inflammation level, and effectiveness of ceftazidime or subtherapeutic colistin to treat the infections were assessed. RESULTS: Inactivation of AmpG or NagZ significantly attenuated the virulence in terms of mice mortality, bacterial load, and inflammation. When inactivating these genes in the dacB-defective background, the ß-lactam resistance phenotype was abolished, disabling the emergence of ceftazidime-resistant mutants, and restoring ceftazidime for treatment. Subtherapeutic colistin was shown to efficiently clear the infection caused by the recycling-defective strains, likely due to the combined effect with the mice cell-wall- targeting immunity. CONCLUSIONS: This study brings us one step closer to new therapies intended to disable P. aeruginosa AmpC-mediated resistance and dampen its virulence, and strongly support the interest in developing efficient AmpG and/or NagZ inhibitors.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Peptidoglicano/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , beta-Lactamas/administração & dosagem , Animais , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Carga Bacteriana , Ceftazidima/administração & dosagem , Parede Celular/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Análise de Sobrevida , Resultado do Tratamento , VirulênciaRESUMO
Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, ß-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type ß-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.
Assuntos
Enterobacter cloacae , Peptidoglicano , beta-Lactamases/metabolismo , Proteínas de Bactérias/metabolismo , Cefalosporinase/genética , Resistência beta-Lactâmica/genética , Testes de Sensibilidade MicrobianaRESUMO
The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).
Assuntos
Anti-Infecciosos , Infecções por Pseudomonas , alfa-Defensinas , Humanos , Colistina/farmacologia , Muramidase/farmacologia , Peptidoglicano , Meropeném/farmacologia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana MúltiplaRESUMO
Given the growing clinical-epidemiological threat posed by the phenomenon of antibiotic resistance, new therapeutic options are urgently needed, especially against top nosocomial pathogens such as those within the ESKAPE group. In this scenario, research is pushed to explore therapeutic alternatives and, among these, those oriented toward reducing bacterial pathogenic power could pose encouraging options. However, the first step in developing these antivirulence weapons is to find weak points in the bacterial biology to be attacked with the goal of dampening pathogenesis. In this regard, during the last decades some studies have directly/indirectly suggested that certain soluble peptidoglycan-derived fragments display virulence-regulatory capacities, likely through similar mechanisms to those followed to regulate the production of several ß-lactamases: binding to specific transcriptional regulators and/or sensing/activation of two-component systems. These data suggest the existence of intra- and also intercellular peptidoglycan-derived signaling capable of impacting bacterial behavior, and hence likely exploitable from the therapeutic perspective. Using the well-known phenomenon of peptidoglycan metabolism-linked ß-lactamase regulation as a starting point, we gather and integrate the studies connecting soluble peptidoglycan sensing with fitness/virulence regulation in Gram-negatives, dissecting the gaps in current knowledge that need filling to enable potential therapeutic strategy development, a topic which is also finally discussed.
Assuntos
Peptidoglicano , beta-Lactamases , Peptidoglicano/metabolismo , Virulência , beta-Lactamases/metabolismo , Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Pseudomonas aeruginosa is a major cause of life-threatening acute infections and life-long lasting chronic infections. The characteristic biofilm mode of life in P. aeruginosa chronic infections severely limits the efficacy of antimicrobial therapies, as it leads to intrinsic tolerance, involving physical and physiological factors in addition to biofilm-specific genes that can confer a transient protection against antibiotics promoting the development of resistance. Indeed, a striking feature of this pathogen is the extraordinary capacity to develop resistance to nearly all available antibiotics through the selection of chromosomal mutations, evidenced by its outstanding and versatile mutational resistome. This threat is dramatically amplified in chronic infections, driven by the frequent emergence of mutator variants with enhanced spontaneous mutation rates. Thus, this mini review is focused on describing the complex interplay of antibiotic resistance mechanisms in P. aeruginosa biofilms, to provide potentially useful information for the design of effective therapeutic strategies.
RESUMO
The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens.
RESUMO
Infectious diseases are still a cause of high mortality and morbidity rates. Current microbiological diagnostic methods are based on culture and phenotypic identification of isolated microorganisms, which can be obtained in about 24-48 h. Given that the microbiological identification is of major importance for patient management, new diagnostic methods are needed in order to detect and identify microorganisms in a timely and accurate manner. Over the last few years, several molecular techniques based on the amplification of microbial nucleic acids have been developed with the aim of reducing the time needed for the identification of the microorganisms involved in different infectious processes. On the other hand, mass spectrometry has emerged as a rapid and consistent alternative to conventional methods for microorganism identification. This review describes the most widely used mass spectrometry technologies -matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization time-of-flight (ESI-TOF)-, both for protein and nucleic acid analysis, as well as the commercial platforms available. Related publications of most interest in clinical microbiology are also reviewed.
Assuntos
Técnicas de Laboratório Clínico/métodos , Espectrometria de Massas/métodos , Técnicas de Tipagem Bacteriana/métodos , Células/química , Serviços de Laboratório Clínico , Técnicas de Laboratório Clínico/instrumentação , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Humanos , Espectrometria de Massas/instrumentação , Técnicas de Tipagem Micológica/métodos , Ácidos Nucleicos/análise , Proteínas/análise , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Virologia/métodosRESUMO
Mammalian innate immunity employs several humoral 'weapons' that target the bacterial envelope. The threats posed by the multidrug-resistant 'ESKAPE' Gram-negative pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are forcing researchers to explore new therapeutic options, including the use of these immune elements. Here we review bacterial envelope-targeting (peptidoglycan and/or membrane-targeting) proteins/peptides of the mammalian immune system that are most likely to have therapeutic applications. Firstly we discuss their general features and protective activity against ESKAPE Gram-negatives in the host. We then gather, integrate, and discuss recent research on experimental therapeutics harnessing their bactericidal power, based on their exogenous administration and also on the discovery of bacterial and/or host targets that improve the performance of this endogenous immunity, as a novel therapeutic concept. We identify weak points and knowledge gaps in current research in this field and suggest areas for future work to obtain successful envelope-targeting therapeutic options to tackle the challenge of antimicrobial resistance.
Assuntos
Acinetobacter baumannii , Animais , Antibacterianos/farmacologia , Mamíferos , Peptídeos , Pseudomonas aeruginosaRESUMO
In the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between ß-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal ß-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other ß-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned ß-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of ß-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers' interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable ß-lactamases, among which the production of OXA-2-derived extended-spectrum ß-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D ß-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired ß-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.
Assuntos
Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Cefalosporinase , Transferência Genética Horizontal , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Mariposas , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Virulência/efeitos dos fármacos , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genéticaRESUMO
Macrolide and fluoroquinolone resistance (MLr/FQr) in Mycoplasma genitalium (MG) infections is concerning worldwide. Current guidelines recommend performing MLr detection in MG-positive cases to adjust antimicrobial therapy. We aimed to evaluate the usefulness of PCR followed by pyrosequencing for MLr detection in comparison with a one-step commercial assay and to assess the prevalence of MLr and FQr in Badalona, Spain. A total of 415 MG-positive samples by Allplex STI-7 (Seegene) were analyzed for MLr detection by pyrosequencing. From those, 179 samples were further analyzed for MG and MLr by ResistancePlus® MG kit (SpeeDx) and 100 of them also for fluoroquinolone resistance (FQr) by sequencing the parC gene. Regarding MG detection, Allplex and Resistance Plus® showed an overall agreement of 87%, but this value rose to 95.4% if we compare them for MLr detection. Prevalence of MLr was 23.1% in Badalona, but this rate increased to 73.7% in the HIV-positive patients cohort. FQr detection showed 3% of resistant strains. Pyrosequencing is a convenient and cheap technique for MLr detection, but one-step tools should be considered in high-throughput laboratories. Despite the fact that MLr remained moderate and FQr was low in our study, simultaneous MG and MLr detection would improve patient's management applying resistance-guided treatment strategies.
RESUMO
In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.
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Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Inibidores de beta-Lactamases/metabolismo , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/uso terapêutico , Cefalosporinase/metabolismo , Cefalosporinase/farmacologia , Cefalosporinase/uso terapêutico , Peptidoglicano/metabolismo , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , Tazobactam/metabolismo , Tazobactam/farmacologia , Tazobactam/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Combinação de Medicamentos , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
The hepatitis C virus (HCV) has been classified into six genotypes and more than 70 subtypes with distinct geographical and epidemiological distributions. While 18 genotype 2 subtypes have been proposed, only 5 have had their complete sequence determined. The aim of this study was to characterize HCV isolates from three patients from the Barcelona metropolitan area of Spain for whom commercial genotyping methods provided discordant results. Full-length genome sequencing was carried out for 2 of the 3 patients; for the third patient only partial NS5B sequences could be obtained. The generated sequences were subjected to phylogenetic, recombination, and identity analyses. Sequences covering most of the HCV genome (9398 and 9566 nt in length) were obtained and showed a 90.3% identity to each other at the nucleotide level, while both sequences differed by 17.5-22.6% from the other fully sequenced genotype 2 subtypes. No evidence of recombination was found. The NS5B phylogenetic tree showed that sequences from the three patients cluster together with the only representative sequence of the provisionally designed 2q subtype, which also corresponds to a patient from Barcelona. Phylogenetic analysis of the full coding sequence showed that subtype 2q was more closely related to subtype 2k. The results obtained in this study suggest that subtype 2q now meets the requirements for confirmed designation status according to consensus criteria for HCV classification and nomenclature, and its epidemiological value is ensured as it has spread among several patients in the Barcelona metropolitan area.
Assuntos
Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/transmissão , Idoso , Análise por Conglomerados , Feminino , Genoma Viral , Genótipo , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Espanha/epidemiologia , Proteínas não Estruturais Virais/genéticaRESUMO
Skin offers protection against external insults, with the skin microbiota playing a crucial defensive role against pathogens that gain access when the skin barrier is breached. Linkages between skin microbes, biofilms and disease have not been well established although single-species biofilm formation by skin microbiota in vitro has been extensively studied. Consequently, the purpose of this work was to optimize and validate a simple polymicrobial biofilm keratinocyte model for investigating commensal, pathogen and keratinocyte interactions and for evaluating therapeutic agents or health promoting interventions. The model incorporates the commensals (Staphylococcus epidermidis and Micrococcus luteus) and pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) which form robust polymicrobial biofilms on immortalized keratinocytes (HaCat cells). We observed that the commensals reduce the damage caused to the keratinocyte monolayer by either pathogen. When the commensals were combined with P. aeruginosa and S. aureus, much thinner biofilms were observed than those formed by the pathogens alone. When P. aeruginosa was inoculated with S. epidermidis in the presence or absence of M. luteus, the commensals formed a layer between the keratinocytes and pathogen. Although S. aureus completely inhibited the growth of M. luteus in dual-species biofilms, inclusion of S. epidermidis in triple or quadruple species biofilms, enabled M. luteus to retain viability. Using this polymicrobial biofilm keratinocyte model, we demonstrate that a quorum sensing (QS) deficient S. aureus agr mutant, in contrast to the parent, failed to damage the keratinocyte monolayer unless supplied with the exogenous cognate autoinducing peptide. In addition, we show that treatment of the polymicrobial keratinocyte model with nanoparticles containing an inhibitor of the PQS QS system reduced biofilm thickness and P. aeruginosa localization in mono- and polymicrobial biofilms.
RESUMO
Many debilitating infections result from persistent microbial biofilms that do not respond to conventional antibiotic regimens. A potential method to treat such chronic infections is to combine agents which interfere with bacterial biofilm development together with an antibiotic in a single formulation. Here, we explore the use of a new bioresponsive polymer formulation derived from specifically modified alginate nanoparticles (NPs) in order to deliver ciprofloxacin (CIP) in combination with the quorum sensing inhibitor (QSI) 3-amino-7-chloro-2-nonylquinazolin-4(3H)-one (ACNQ) to mature Pseudomonas aeruginosa biofilms. The alginate NPs were engineered to incorporate a pH-responsive linker between the polysaccharide backbone and the QSI, and to encapsulate CIP via charge-charge interactions of the positively-charged drug with the carboxyl residues of the alginate matrix. In this way, a dual-action release of antibiotic and QSI was designed for the low-pH regions of a biofilm, involving cleavage of the QSI-linker to the alginate matrix and reduced charge-charge interactions between CIP and the polysaccharide as the alginate carboxyl side-chains protonated. When tested in a biofilm model the concomitant release of CIP + QSI from the pH-responsive nanoparticles significantly reduced the viability of the biofilm compared with CIP treatment alone. In addition, the alginate NPs were shown to penetrate deeply into P. aeruginosa biofilms, which we attribute in part to the charges of the NPs and the release of the QSI agent. Finally, we tested the formulation in both a 2D keratinocyte and a 3D ex vivo skin infection model. The dual-action bio-responsive QSI and CIP release nanoparticles effectively cleared the infection in the latter, suggesting considerable promise for combination therapeutics which prevent biofilm formation as well as effectively killing mature P. aeruginosa biofilms.
Assuntos
Ciprofloxacina/uso terapêutico , Nanopartículas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Linhagem Celular , Ciprofloxacina/química , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/patogenicidade , SuínosRESUMO
OBJECTIVES: Onco-haematological patients are prone to develop infections, and antibiotic prophylaxis may lead to negative blood cultures. Thus, the microbiological diagnosis and subsequent administration of a targeted antimicrobial therapy is often difficult. The goal of this study was to evaluate the usefulness of IRIDICA (PCR/ESI-MS technology) for the molecular diagnosis of bloodstream infections in this patient group. METHODS: A total of 463 whole blood specimens from different sepsis episodes in 429 patients were analysed using the PCR/ESI-MS platform, comparing the results with those of blood culture and other clinically relevant information. RESULTS: The sensitivity of PCR/ESI-MS by specimen (excluding polymicrobial infections, n = 25) in comparison with blood culture was 64.3% overall, 69.0% in oncological patients, and 59.3% in haematological patients. When comparing with a clinical infection criterion, overall sensitivity rose to 74.7%, being higher in oncological patients (80.0%) than in haematological patients (67.7%). Thirty-one microorganisms isolated by culture were not detected by IRIDICA, whereas 42 clinically relevant pathogens not isolated by culture were detected moleculary. CONCLUSIONS: PCR/ESI-MS offers a reliable identification of pathogens directly from whole blood. While additional studies are needed to confirm our findings, the system showed a lower sensitivity in onco-haematological patients in comparison with previously reported results in patients from the Intensive Care Unit.
Assuntos
Neoplasias Hematológicas/complicações , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase , Sepse/diagnóstico , Espectrometria de Massas por Ionização por Electrospray , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/complicações , Bacteriemia/diagnóstico , Bacteriemia/microbiologia , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Sensibilidade e Especificidade , Sepse/complicações , Sepse/microbiologia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto JovemRESUMO
BACKGROUND: Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient's blood. METHODS: A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). RESULTS: The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. CONCLUSIONS: IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient's blood within 6h, which brings the opportunity to improve management of septic patients, especially for those critically ill admitted to the ICU.