RESUMO
The crisis of bacterial resistance is an emerging One Health challenge, driven by the overuse of antimicrobials in medical and agricultural settings. This study aimed to investigate extended-spectrum ß-lactamase (ESBL), Ampicillinase (AmpC), and carbapenemase production, and the presence of genes encoding these enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., major contributors to infections and resistance isolates from animals. From 2016 to 2021, 130 multidrug-resistant (MDR) or extensively drug-resistant (XDR) isolates were recovered from the secretions, excretions, and organs of companion and production animals with active infections. Antibacterial sensitivity tests, along with phenotypic and genotypic detection of resistance enzymes, were performed. To the best of our knowledge, this is the first study in Brazil to estimate the prevalence of XDR Enterobacteriales isolated from companion and production animals, which accounted for 13.8% of the strains. Statistically significant differences (P < 0.05) in resistant bacteria between different classes and within the same class of antibacterial bacteria were found. The statistical probability between genotypic detection of ESBL (OR = 3.1) and phenotypic tests for AmpC (OR = 2.3) was also established. Approximately 32.3%, 17.6%, and 16.8% of the strains had positive phenotypic tests for ESBL, AmpC, and carbapenemases, respectively. Genetic analysis revealed the presence of blaCTX-M (60.0%), blaAmpC (9.18%), blaKPC-2 (0.76%), and blaNDM (1.52%). AmpC genes were identified in 8.46% of the samples, with blaCMY being the most frequent (6.92%), followed by blaDHA (0.77%), and blaFOX (0.77%). The sequenced amplicons were deposited in NCBI. This study reveals critical data on Enterobacteriaceae with antibacterial resistance genes isolated from animals and may pose a significant threat to One health.
Assuntos
Antibacterianos , Proteínas de Bactérias , Infecções por Enterobacteriaceae , Enterobacteriaceae , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Animais , Brasil , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/veterinária , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Plasmídeos/genética , Farmacorresistência Bacteriana Múltipla/genética , Animais de Estimação/microbiologia , Fenótipo , Genótipo , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologiaRESUMO
Introduction. In critically ill patients, the occurrence of multidrug-resistant Pseudomonas aeruginosa infection is a significant concern, given its ability to acquire multidrug-resistant, form biofilms and secrete toxic effectors.Hypothesis or Gap Statement. In Brazil, limited data are available regarding the prevalence of dissemination, and the impact of the type III secretion system (T3SS) on toxin production and biofilm formation in clinical isolates of P. aeruginosa.Aim. This study investigates the dissemination of virulent P. aeruginosa harbouring the bla KPC-2 and bla PDC-5 genes, the presence of T3SS genes and their biofilm-forming capability.Methodology. A total of 128 non-duplicate clinical isolates of carbapenem-resistant P. aeruginosa (CRPA) from different sources collected from eight hospitals were examined. Detection was performed by PCR of the T3SS genes (exoU, exoT, exoS and exoY), carbapenemases (bla KPC, bla GIM and bla NDM) and beta-lactamase gene (bla PDC). PFGE and phenotypic biofilm production (initial adhesion assay and biofilm cell concentration) were performed.Results. We found exoT+ (86%) to be the most frequent genotypic variant, followed by exoY+ (61%). Notably, a substantial proportion of isolates exhibited the simultaneous presence of exoU+ and exoS+ genes, along with a high prevalence of bla KPC-2 + (64%) and bla PDC-5 + (64%) among the disseminated clones in the evaluated region. Additionally, 78% of the isolates demonstrated biofilm-forming capability, and two distinct clonal profiles were identified and disseminated both intra- and inter-hospital. Also, it was revealed that the exoU genotype was significantly more frequent among multidrug-resistant strains.Conclusion. These findings underscore the ability of multiple virulent and biofilm-producing clones of CRPA to propagate effectively.
Assuntos
Proteínas de Bactérias , Biofilmes , Infecções por Pseudomonas , Pseudomonas aeruginosa , Sistemas de Secreção Tipo III , beta-Lactamases , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/fisiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Infecções por Pseudomonas/microbiologia , Brasil , Hospitais , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Virulência/genética , GenótipoRESUMO
Antimicrobial resistance (AMR) is a major threat to global public health that continues to grow owing to selective pressure caused by the use and overuse of antimicrobial drugs. Resistance spread by plasmids is of special concern, as they can mediate a wide distribution of AMR genes, including those encoding extended-spectrum ß-lactamases (ESBLs). The CTX-M family of ESBLs has rapidly spread worldwide, playing a large role in the declining effectiveness of third-generation cephalosporins. This rapid spread across the planet is puzzling given that plasmids carrying AMR genes have been hypothesized to incur a fitness cost to their hosts in the absence of antibiotics. Here, we focus on a WT plasmid that carries the bla CTX-M 55 ESBL gene. We examine its conjugation rates and use head-to-head competitions to assay its associated fitness costs in both laboratory and wild Escherichia coli strains. We found that the wild strains exhibit intermediate conjugation levels, falling between two high-conjugation and two low-conjugation laboratory strains, the latter being older and more ancestral. We also show that the plasmid increases the fitness of both WT and lab strains when grown in lysogeny broth and Davis-Mingioli media without antibiotics, which might stem from metabolic benefits conferred on the host, or from interactions between the host and the rifampicin-resistant mutation we used as a selective marker. Laboratory strains displayed higher conjugation frequencies compared to WT strains. The exception was a low-passage K-12 strain, suggesting that prolonged laboratory cultivation may have compromised bacterial defences against plasmids. Despite low transfer rates among WT E. coli, the plasmid carried low fitness cost in minimal medium but conferred improved fitness in enriched medium, indicating a complex interplay between plasmids, host genetics and environmental conditions. Our findings reveal an intricate relationship between plasmid carriage and bacterial fitness. Moreover, they show that resistance plasmids can confer adaptive advantages to their hosts beyond AMR. Altogether, these results highlight that a closer study of plasmid dynamics is critical for developing a secure understanding of how they evolve and affect bacterial adaptability that is necessary for combating resistance spread.
Assuntos
Escherichia coli , Aptidão Genética , Plasmídeos , beta-Lactamases , Plasmídeos/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacologia , Conjugação GenéticaRESUMO
Carbapenemase-producing Enterobacterales are a growing concern in public health. In order to rapidly determine the antimicrobial profile, the MALDI Biotyper - antibiotic susceptibility test rapid assay (MBT-ASTRA) was developed, based on the relative growth of bacteria in the presence of antibiotics. In this study, we added carbapenemase enzymatic inhibitors to the MBT-ASTRA and developed an adapted method named MALDI Biotyper - Phenotypic Identification Test Rapid Assay (MBT-PITRA) in order to perform a rapid and cost-effective phenotypic test to detect Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM), including co-producers, in Enterobacterales. Fifty-nine clinical isolates of carbapenemase-producing Enterobacterales and 28 non-carbapenemase-producing isolates collected from 2013 to 2023 were assessed using this approach. The MBT-PITRA method involved incubating bacterial isolates with different solutions of meropenem plus enzymatic inhibitors, phenylboronic acid and/or ethylenediaminetetraacetic acid, followed by analysis using MALDI-TOF MS. Carbapenemase production was inferred based on relative growth (RG) values calculated from peak intensities in the presence and absence of enzymatic inhibitors. KPC-producing isolates presented 90 % (18/20) concordance, while isolates positive for NDM (16/16), KPC plus NDM (14/14) and negative (28/28) for carbapenemases were 100 % correctly identified. MBT-PITRA demonstrated to be a promising method for identification of carbapenemases. IMPORTANCE: The MBT-PITRA appears to be a promising method for the rapid detection of carbapenemases in Enterobacterales.
Assuntos
Antibacterianos , Proteínas de Bactérias , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases , beta-Lactamases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas de Bactérias/metabolismo , Humanos , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/efeitos dos fármacos , Fenótipo , Meropeném/farmacologia , Infecções por Enterobacteriaceae/microbiologiaRESUMO
Wastewater serves as a reservoir for antimicrobial-resistant bacteria. This study revealed the presence of carbapenem-resistant and carbapenemase-producing Gram-negative bacilli (GNB), established clonal relationships among isolates in hospital and municipal wastewater, and identified a high-risk clone in municipal wastewater. A total of 63 isolates of GNB were obtained, with Enterobacterales being the most frequently isolated group (62%). Carbapenemase-producing Lelliottia amnigena, Kluyvera cryocrescens, and Shewanella putrefaciens isolates were documented for the first time in Mexico. The detectableted carbapenemase genes were blaKPC (55%), blaNDM (12%), blaVIM-2 (12%), blaOXA-48 (4%), blaGES (2%), blaNDM-1 (2%), and blaNDM-5 (2%). Clonal relationships were observed among Klebsiella pneumoniae and Enterobacter spp. isolates, and remarkably the high-risk clone Escherichia coli ST361, carrying blaNDM-5, was identified. This study demonstrates that wastewater harbours carbapenem-resistant and carbapenemase-producing bacteria, posing a public health threat that requires epidemiological surveillance.
Assuntos
Proteínas de Bactérias , Bactérias Gram-Negativas , Hospitais , Águas Residuárias , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Águas Residuárias/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , México , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Testes de Sensibilidade Microbiana , Humanos , Antibacterianos/farmacologiaRESUMO
The rise of antibiotic-resistant bacteria in clinical settings has become a significant global concern. Among these bacteria, Acinetobacter baumannii stands out due to its remarkable ability to acquire resistance genes and persist in hospital environments, leading to some of the most challenging infections. Horizontal gene transfer (HGT) plays a crucial role in the evolution of this pathogen. The A. baumannii AMA205 strain, belonging to sequence type ST79, was isolated from a COVID-19 patient in Argentina in 2021. This strain's antimicrobial resistance profile is notable as it harbors multiple resistance genes, some of which had not been previously described in this species. The AmpC family ß-lactamase blaCMY-6, commonly found in Enterobacterales, had never been detected in A. baumannii before. Furthermore, this is the first ST79 strain known to carry the carbapenemase blaNDM-1 gene. Other acquired resistance genes include the carbapenemase blaOXA-23, further complicating treatment. Susceptibility testing revealed high resistance to most antibiotic families, including cefiderocol, with significant contributions from blaCMY-6 and blaNDM-1 genes to the cephalosporin and carbapenem resistance profiles. The A. baumannii AMA205 genome also contains genetic traits coding for 111 potential virulence factors, such as the iron-uptake system and biofilm-associated proteins. This study underscores A. baumannii's ability to acquire multiple resistance genes and highlights the need for alternative therapies and effective antimicrobial stewardship to control the spread of these highly resistant strains.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , beta-Lactamases , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Humanos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Antibacterianos/farmacologia , COVID-19/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Transferência Genética Horizontal , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Genoma Bacteriano , Argentina , Fatores de Virulência/genéticaRESUMO
Antibiotic resistance (ABR) is a critical and growing global challenge, especially in low- and middle-income countries. Ecuador has made great progress in connecting households to piped water supplies; however, the collection and treatment of domestic wastewater has lagged. This infrastructural gap may be accelerating the spread of ABR into surface waters used downstream for irrigation. We studied the contributions of a small town in Ecuador to the prevalence of extended-spectrum ß-lactamase-producing Escherichia coli in a glacial stream used for irrigating crops. The study analyzed water samples upstream (n = 60) and downstream (n = 60) of the town of Píntag as well as 30 lettuce samples irrigated by surface waters downstream of the town. A subset of third generation cephalosporin resistant E. coli (3GCR-EC) isolates (n = 58) were sequenced to characterize antibiotic resistance genes and pathogenic lineages. Our results showed that there was nearly a three-log increase in mean E. coli colony forming units in the downstream samples versus upstream. At the upstream sites above the town of Píntag, 6.7% of water samples were positive for 3GCR-EC compared to 100% of samples collected at the downstream sites. Additionally, 70.1% of sequenced 3GCR-EC isolates collected at downstream sites carried blaCTX-M genes and 3.4% belonged to pandemic lineages ST131 and ST10. As countries develop household piped water infrastructure, attention should focus on how the lack of domestic wastewater collection and treatment may accelerate the spread of ABR in waterways and the food system.
Assuntos
Irrigação Agrícola , Escherichia coli , Águas Residuárias , beta-Lactamases , Águas Residuárias/microbiologia , beta-Lactamases/metabolismo , Equador , Microbiologia da Água , Eliminação de Resíduos Líquidos/métodos , Purificação da Água/métodos , Antibacterianos/farmacologiaRESUMO
Currently, antimicrobial resistance (AMR) is a serious health problem in the world, mainly because of the rapid spread of multidrug-resistant (MDR) bacteria. These include bacteria that produce ß-lactamases, which confer resistance to ß-lactams, the antibiotics with the most prescriptions in the world. Carbapenems are particularly noteworthy because they are considered the ultimate therapeutic option for MDR bacteria. However, this group of antibiotics can also be hydrolyzed by ß-lactamases, including metallo-ß-lactamases (MBLs), which have one or two zinc ions (Zn2+) on the active site and are resistant to common inhibitors of serine ß-lactamases, such as clavulanic acid, sulbactam, tazobactam, and avibactam. Therefore, the design of inhibitors against MBLs has been directed toward various compounds, with groups such as nitrogen, thiols, and metal-binding carboxylates, or compounds such as bicyclic boronates that mimic hydrolysis intermediates. Other compounds, such as dipicolinic acid and aspergillomarasmin A, have also been shown to inhibit MBLs by chelating Zn2+. In fact, recent inhibitors are based on Zn2+ chelation, which is an important factor in the mechanism of action of most MBL inhibitors. Therefore, in this review, we analyzed the current strategies for the design and mechanism of action of metal-ion-binding inhibitors that combat MDR bacteria.
Assuntos
Zinco , Inibidores de beta-Lactamases , beta-Lactamases , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , beta-Lactamases/química , Zinco/química , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Metais/química , Bactérias/efeitos dos fármacos , Bactérias/enzimologiaRESUMO
Klebsiella pneumoniae strains that produce Klebsiella pneumoniae Carbapenemase (KPC) variants displaying resistance to ceftazidime-avibactam (CZA) often remain susceptible to meropenem (MEM), suggesting a potential therapeutic use of this carbapenem antibiotic. However, in vitro studies indicate that these sorts of strains can mutate becoming MEM-resistant, raising concerns about the effectiveness of carbapenems as treatment option. We have studied mutation rates occurring from the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae belonging to ST11. Two-step fluctuation assays (FAs) were conducted. In brief, initial cultures of KPC-114-producing K. pneumoniae showing 1 µg/mL MEM MIC were spread on Mueller-Hinton agar plates containing 2-8 µg/mL MEM. A second step of FA, at 4-16 µg/mL MEM was performed from a mutant colony obtained at 2 µg/mL MEM. Mutation rates were calculated using maximum likelihood estimation. Parental and mutant strains were sequenced by Illumina NextSeq, and mutations were predicted by variant-calling analysis. At 8 µg/mL MEM, mutants derived from parental CZA-resistant (MIC ≥ 64 µg/mL)/MEM-susceptible (MIC = 1 µg/mL) KPC-114-positive K. pneumoniae exhibited an accumulative mutation rate of 3.05 × 10-19 mutations/cell/generation, whereas at 16 µg/mL MEM an accumulative mutation rate of 1.33 × 10-19 mutations/cell/generation resulted in the reversion of KPC-114 (S181_P182 deletion) to KPC-2. These findings highlight that the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae ST11 is related to low mutation rates suggesting a low risk of therapeutic failure. In vivo investigations are necessary to confirm the clinical potential of MEM against CZA-resistant KPC variants.IMPORTANCEThe emergence of ceftazidime-avibactam (CZA) resistance among carbapenem-resistant Klebsiella pneumoniae is a major concern due to the limited therapeutic options. Strikingly, KPC mutations mediating CZA resistance are generally associated with meropenem susceptibility, suggesting a potential therapeutic use of this carbapenem antibiotic. However, the reversion of meropenem-susceptible to meropenem-resistant could be expected. Therefore, knowing the mutation rate related to this genetic event is essential to estimate the potential use of meropenem against CZA-resistant KPC-producing K. pneumoniae. In this study, we demonstrate, in vitro, that under high concentrations of meropenem, reversion of KPC-114 to KPC-2 in CZA-resistant/meropenem-susceptible K. pneumoniae belonging to the global high-risk ST11 is related to low mutation rates.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Proteínas de Bactérias , Ceftazidima , Combinação de Medicamentos , Infecções por Klebsiella , Klebsiella pneumoniae , Meropeném , Testes de Sensibilidade Microbiana , Taxa de Mutação , beta-Lactamases , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Ceftazidima/farmacologia , Compostos Azabicíclicos/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meropeném/farmacologia , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , MutaçãoRESUMO
Staphylococcus aureus is a bacterial pathogen that causes bloodstream infections, pneumonia, and skin abscesses and is the primary pathogen responsible for medical devices associated with biofilm infections, accounting for approximately 70 % of cases. Therefore, the World Health Organization (WHO) has designated this microorganism as a top priority due to its role in causing over 20,000 bacteremia-related deaths in the US each year. The issue of pathogen resistance to antibiotics, mainly by a biofilm, further complicates these infections since biofilms render the bacterial colony impervious to antibiotics. However, many natural and synthetic substances also induce bacterial biofilm formation. Therefore, we investigated whether the most common active pharmaceutical ingredients (APIs) could induce biofilm formation in two clinical isolates of extended-spectrum beta-lactamase Staphylococcus aureus, one of them also methicillin-resistant (A2M) and two medical devices. We detected biofilm inducers, inhibitors, and destabilizers. Microbial strain, medical devices, API structure, and concentration influenced the modulatory effects of biofilm. In all devices tested, including microplates, FR18 duodenal probe, and respiratory probe, the clinic isolate methicillin-resistant S. aureus A2M exhibited lower susceptibility to biofilm formation than S. aureus A1. The anti-inflammatory acetaminophen, the hypocholesterolemic lovastatin, and the diuretic hydrochlorothiazide all induced biofilm. However, verapamil, an antihypertensive, and cetirizine, an antihistamine, inhibited biofilm on S. aureus A2M, while propranolol, another antihypertensive, inhibited biofilm on S. aureus A1. Additionally, diclofenac, an analgesic, and cetirizine destabilized the biofilm, resulting in more free bacteria and possibly making them more susceptible to external agents such as antibiotics. Nonetheless, further epidemiologic analyses and in vivo assays are needed to confirm these findings and to establish a correlation between drug use, the onset of bacterial infections in patients, and the use of medical devices. This work provides information about the probable clinical implications of drugs in patients using medical devices or undergoing surgical procedures. Inhibitory APIs could also be used as drug repurposing or templates to design new, more potent biofilm inhibitors.
Assuntos
Antibacterianos , Biofilmes , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas , Staphylococcus aureus , beta-Lactamases , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Antibacterianos/farmacologia , Humanos , Staphylococcus aureus/efeitos dos fármacos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamases/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacosRESUMO
Antimicrobial resistance and biofilm formation by microbial pathogens pose a significant challenge to poultry production systems due to the persistent risk of dissemination and compromise of bird health and productivity. In this context, the study aimed to investigate the occurrence of different multiresistance phenotypes and the biofilm-forming ability of Enterobacteriaceae isolated from broiler chicken excreta in poultry production units in Ceará, Brazil. Samples were collected from three distinct broiler breeding facilities and subjected to isolation, identification, antibiotic susceptibility testing, phenotypic screening for ß-lactamases enzymes, and biofilm formation evaluation. Seventy-one strains were identified, being Escherichia coli (37 %) and Proteus mirabilis (32 %), followed by Klebsiella pneumoniae (11 %), Providencia stuartii (9 %), Klebsiella aerogenes (6 %), Alcaligenes faecalis (4 %), and Salmonella sp. (1 %). A significant proportion (87 %) of multiresistant strains were detected. For the phenotypic evaluation of ß-lactamases production, strains with resistance to second and third-generation cephalosporins and carbapenems were tested. About 4 of 6 and 10 of 26 were positive for inducible chromosomal AmpC ß-lactamase and extended-spectrum ß-lactamase (ESBL), respectively. Regarding biofilm formation, it was observed that all MDR strains were capable of forming biofilm. In this sense the potential of these MDR bacteria to develop biofilms becomes a significant concern, representing a real threat to both human and animal health, as biofilms offer stability, antimicrobial protection, and facilitate genetic transfer.
Assuntos
Antibacterianos , Biofilmes , Galinhas , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae , Fazendas , Fezes , Testes de Sensibilidade Microbiana , beta-Lactamases , Animais , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Brasil , beta-Lactamases/genética , beta-Lactamases/metabolismo , Fezes/microbiologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/genética , Antibacterianos/farmacologia , Galinhas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Aves Domésticas/microbiologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/veterináriaRESUMO
The use of ß-lactam/ß-lactamase inhibitors constitutes an important strategy to counteract ß-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A ß-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other ß-lactamases.
Assuntos
Compostos Azabicíclicos , Inibidores de beta-Lactamases , beta-Lactamases , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/química , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Cristalografia por Raios X , Antibacterianos/farmacologia , Imipenem/farmacologia , Imipenem/química , Ceftazidima/farmacologia , Testes de Sensibilidade Microbiana , Domínio CatalíticoRESUMO
The bacterium Escherichia coli is one of the main causes of urinary tract infections. The formation of bacterial biofilms, especially associated with the use of urinary catheters, contributes to the establishment of recurrent infections and the development of resistance to treatment. Strains of E. coli that produce extended-spectrum beta-lactamases (ESBL) have a greater ability to form biofilms. In addition, there is a lack of drugs available in the market with antibiofilm activity. Promethazine (PMZ) is an antihistamine known to have antimicrobial activity against different pathogens, including in the form of biofilms, but there are still few studies of its activity against ESBL E. coli biofilms. The aim of this study was to evaluate the antimicrobial activity of PMZ against ESBL E. coli biofilms, as well as to assess the application of this drug as a biofilm prevention agent in urinary catheters. To this end, the minimum inhibitory concentration and minimum bactericidal concentration of PMZ in ESBL E. coli strains were determined using the broth microdilution assay and tolerance level measurement. The activity of PMZ against the cell viability of the in vitro biofilm formation of ESBL E. coli was analyzed by the MTT colorimetric assay and its ability to prevent biofilm formation when impregnated in a urinary catheter was investigated by counting colony-forming units (CFU) and confirmed by scanning electron microscopy (SEM). PMZ showed bactericidal activity and significantly reduced (p < 0.05) the viability of the biofilm being formed by ESBL E. coli at concentrations of 256 and 512 µg/ml, as well as preventing the formation of biofilm on urinary catheters at concentrations starting at 512 µg/ml by reducing the number of CFUs, as also observed by SEM. Thus, PMZ is a promising candidate to prevent the formation of ESBL E. coli biofilms on abiotic surfaces.
Assuntos
Antibacterianos , Biofilmes , Escherichia coli , Testes de Sensibilidade Microbiana , Prometazina , Cateteres Urinários , beta-Lactamases , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Prometazina/farmacologia , Escherichia coli/efeitos dos fármacos , beta-Lactamases/metabolismo , Cateteres Urinários/microbiologia , Antibacterianos/farmacologia , Humanos , Infecções Urinárias/microbiologia , Viabilidade Microbiana/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológicoRESUMO
Resistance to carbapenems emerged in clinical settings and has rapidly spread to other sectors, such as food and the environment, representing a One Health problem. In this regard, vegetables contaminated by critical priority pathogens have raised global concerns. Here, we have performed a whole-genome sequence-based analysis of extensively drug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa strains isolated from cabbage, spinach, and lettuce, respectively. Genomic analysis revealed the emergence of international and high-risk clones belonging to ST340, ST155, and ST233, harboring a broad resistome to clinically important antimicrobials. In this context, K. pneumoniae, E. coli, and P. aeruginosa strains carried blaKPC-2, blaNDM-1, and blaVIM-2, respectively. The blaKPC-2 gene with a non-Tn4401 element (NTEKPC-Ic) was located on an IncX3-IncU plasmid, while the blaVIM-2 gene was associated with a Tn402-like class 1 integron, In559, on the chromosome. Curiously, the blaNDM-1 gene coexisted with the blaPER-2 gene on an IncC plasmid and the regions harboring both genes contained sequences of Tn3-like element ISKox2-like family transposase. Comparative genomic analysis showed interspecies and clonal transmission of carbapenemase-encoding genes at the human-animal-environmental interface. These findings raise a food safety alert about hospital-associated carbapenemase producers, supporting that fresh vegetables can act as a vehicle for the spread of high-risk clones.
Assuntos
Verduras , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Verduras/microbiologia , Inocuidade dos Alimentos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Microbiologia de Alimentos , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Sequenciamento Completo do Genoma , HumanosRESUMO
Multidrug-resistant pathogenic vibrios are a crisis of concern as they cause multiple illnesses, including gastroenteritis in humans and acute hepatopancreatic necrosis in aquaculture. In the current study, we investigated the prevalence of the beta-lactamase gene CTX-M-group 1 in Vibrio spp. (Vibrio cholerae and Vibrio parahaemolyticus) from the water and sediment of urban tropical mangrove ecosystems of Kerala, southwest India. A total of 120 isolates of Vibrio spp. were tested for antibiotic susceptibility to 14 antibiotics. In water, ampicillin resistance was very high in isolates of V. cholerae (94.1%, n = 17) and V. parahaemolyticus (89.1%, n = 46). 26.9% of V. parahaemolyticus and 14.2% of V. cholerae harbored the CTX-M-group 1 gene in water samples. Compared to V. cholerae, the CTX-M-group 1 gene was exclusively hosted by V. parahaemolyticus (49%) in sediment samples. A significant difference in the prevalence of the CTX-M-group 1 gene was observed among Vibrio spp. in both water and sediment samples (p < 0.05). The results revealed the presence of multidrug-resistant and beta-lactamase harboring Vibrio spp. in mangrove ecosystems, which may have evolved as a consequence of the misuse and abuse of broad-spectrum antibiotics as prophylaxis in human health care and aquaculture.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Vibrio cholerae , Vibrio parahaemolyticus , beta-Lactamases , Índia/epidemiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/enzimologia , Vibrio cholerae/isolamento & purificação , Antibacterianos/farmacologia , Ecossistema , Áreas Alagadas , Sedimentos Geológicos/microbiologiaRESUMO
The objective of the study was to evaluate the frequency and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella spp. and the risk factors associated with a high total bacterial count in bulk tank milk samples of dairy farms in three municipalities of the Antioquia Department, Colombia. Fifteen samples were positive for E. coli and Klebsiella spp. Subsequent analysis of the 16 S rRNA gene sequences confirmed these isolates included E. coli (n = 3), K. oxytoca (n = 11), and K. pneumoniae (n = 1). None of the isolates was positive for ESBL identification by phenotypic methods, but the only the isolate of K. pneumoniae was positive for the blaSHV61 gene by sequence analysis. The antibiotic susceptibility evaluation for all Klebsiella spp. isolates identified resistance to fosfomycin (50%; 6/12) and ampicillin (100%; 12/12). While most of the herds maintain adequate hygienic quality, specific risk factors such as having more than 60 milking cows, frequent changes in milkers, milking in paddocks, and using a chlorinated product for pre-dipping have been identified as associated with a high total bacterial count > 100,000 CFU/mL in bulk tank milk. However, certain variables including the milker being the owner of the animals and the proper washing and disinfection of the milking machine contribute to maintain a high level of hygiene and quality in the raw milk stored in the tanks. In conclusion, the frequency of ESBL producers was relatively low, with only K. pneumoniae testing positive for the blaSHV ESBL type. The presence of these bacteria in milk tanks represents a potential risk to public health for consumers of raw milk and its derivatives.
Assuntos
Antibacterianos , Klebsiella pneumoniae , Leite , beta-Lactamases , Animais , Leite/microbiologia , Colômbia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Fatores de Risco , Bovinos , Antibacterianos/farmacologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Carga Bacteriana , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/veterinária , Indústria de Laticínios , Fazendas , FemininoRESUMO
Prompt and precise identification of carbapenemase-producing organisms is crucial for guiding clinical antibiotic treatments and limiting transmission. Here, we propose modifying the Blue Carba test (BCT) and Carba NP-direct (CNPd) to identify molecular carbapenemase classes, including dual carbapenemase strains, by adding specific Class A and Class B inhibitors. We tested 171 carbapenemase-producing Gram-negative bacilli strains-21 in Class A (KPC, NMC, SME), 58 in Class B (IMP, VIM, NDM, SPM), and 92 with dual carbapenemase production (KPC+NDM, KPC+IMP, KPC+VIM), all previously positive with BCT or CNPd. We also included 13 carbapenemase non-producers. ß-lactamases were previously characterized by PCR. The improved BCT/CNPd methods detect imipenem hydrolysis from an imipenem-cilastatin solution, using pH indicators and Class A (avibactam) and/or Class B (EDTA) inhibitors. Results were interpreted visually based on color changes. CNPd achieved 99.4% sensitivity and 100% specificity in categorizing carbapenemases, while BCT had 91.8% sensitivity and 100% specificity. Performance varied by carbapenemase classes: both tests classified all Class A-producing strains. For Class B, the CNP test identified 57/58 strains (98.3%), whereas the BCT test, 45/58 strains (77.6%), with non-fermenters posing the greatest detection challenge. For Classes A plus B dual producers, both tests performed exceptionally well, with only one indeterminate strain for the BCT. The statistical comparison showed both methods had similar times to a positive result, with differences based on the carbapenemase class or bacterial group involved. This improved assay rapidly distinguishes major Class A or Class B carbapenemase producers among Gram-negative bacilli, including dual-class combinations, in less than 2 hours. IMPORTANCE: Rapid and accurate identification of carbapenemase-producing organisms is of vital importance in guiding appropriate clinical antibiotic treatments and curbing their transmission. The emergence of negative bacilli carrying multiple carbapenemase combinations during and after the severe acute respiratory syndrome coronavirus 2 pandemic has posed a challenge to the conventional biochemical tests typically used to determine the specific carbapenemase type in the isolated strains. Several initiatives have aimed to enhance colorimetric methods, enabling them to independently identify the presence of Class A or Class B carbapenemases. Notably, no previous efforts have been made to distinguish both classes simultaneously. Additionally, these modifications have struggled to differentiate between carriers of multiple carbapenemases, a common occurrence in many Latin American countries. In this study, we introduced specific Class A and Class B carbapenemase inhibitors into the Blue Carba test (BCT) and Carba NP-direct (CNP) colorimetric assays to identify the type of carbapenemase, even in cases of multiple carbapenemase producers within these classes. These updated assays demonstrated exceptional sensitivity and specificity (≥ 90%) all within a rapid turnaround time of under 2 hours, typically completed in just 45 minutes. These in-house enhancements to the BCT and CNP assays present a rapid, straightforward, and cost-effective approach to determining the primary carbapenemase classes. They could serve as a viable alternative to molecular biology or immuno-chromatography techniques, acting as an initial diagnostic step in the process.
Assuntos
Antibacterianos , Proteínas de Bactérias , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/análise , beta-Lactamases/metabolismo , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/classificação , Humanos , Antibacterianos/farmacologia , Sensibilidade e Especificidade , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Imipenem/farmacologiaRESUMO
Introduction: Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum ß-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Methods: Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Results: Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum ß-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other ß-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other ß-lactams.
Assuntos
Proteínas de Bactérias , Ceftazidima , Farmacorresistência Bacteriana Múltipla , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Ceftazidima/farmacologia , Chile , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Mutação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/microbiologia , Sequenciamento Completo do GenomaRESUMO
I. paraguariensis St. Hil. is a south American species of agronomic interest with studies supporting its medicinal properties. As the investigation of active ingredients with antimicrobial effect from medicinal plants is a suitable approach to the current antibacterial resistance problem, the aim of the present study was to determine the antibacterial activity of yerba mate ethanolic extracts against carbapenemase-producing gram-negative bacteria (reference strains and clinical isolates). Extracts showed antibacterial activity against Klebsiella pneumoniae ATCC® BAA-2342™ (KPC producing), Providencia rettgeri (NDM producing), Pseudomonas aeruginosa (MBL producing) and P. aeruginosa (VIM producing) at the concentrations tested. The Minimal-Inhibitory-Concentration and Minimal-Bactericidal-Concentration values ranged between 1 and 32 mg.ml-1 for the reference strains, and between 0.125 and 1 mg.ml-1 for the clinical isolates. The MBC/MIC index characterized the extracts as bactericidal. The combinations of commercial antibiotics and extracts showed a synergistic action on the reference strains studied. The lethal concentration 50 obtained using the Artemia salina toxicity assay were higher than 1 mg.ml-1 for all the extracts, indicating a low toxicity. The in vitro activity and low toxicity suggest that ethanolic I. paraguariensis leaf extracts constitute an outstanding source for new antibacterial compounds, and further studies should be carried out to understand their mechanism of action.
Assuntos
Antibacterianos , Proteínas de Bactérias , Bactérias Gram-Negativas , Ilex paraguariensis , Testes de Sensibilidade Microbiana , Extratos Vegetais , Folhas de Planta , beta-Lactamases , Extratos Vegetais/farmacologia , Ilex paraguariensis/química , beta-Lactamases/metabolismo , beta-Lactamases/biossíntese , Folhas de Planta/química , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologiaRESUMO
We describe four cases of a novel carbapenem-resistant Pseudomonas aeruginosa ST179 clone carrying the blaKPC-2 or blaKPC-35 gene together with blaIMP-16, imported from Peru to Spain and isolated from leukemia patients. All isolates were multidrug-resistant but remained susceptible to fosfomycin, cefiderocol, and colistin. Whole-genome sequencing revealed that blaKPC-2 and blaKPC-35 were located in an IncP6 plasmid, whereas blaIMP-16 was in a chromosomal type 1 integron. This study highlights the global threat of multidrug-resistant P. aeruginosa clones and underscores the importance of monitoring and early detection of emerging resistance mechanisms to guide appropriate treatment strategies. The importation and spread of such clones emphasize the urgent need to implement strict infection control measures to prevent the dissemination of carbapenem-resistant bacteria. IMPORTANCE: This is the first documented case of a Pseudomonas aeruginosa ST179 strain carrying the blaKPC-35 gene, and it represents the first report of a P. aeruginosa co-harboring blaIMP-16 and either blaKPC-2 or blaKPC-35, which wre imported from Peru to Spain, highlighting a threat due to the capacity of spreading carbapenem-resistance via plasmid conjugation.