Ceftazidime/Tobramycin Co-Loaded Chitosan-Coated Zein Nanoparticles against Antibiotic-Resistant and Biofilm-Producing Pseudomonas aeruginosa and Klebsiella pneumoniae.

This study aimed to co-encapsulate ceftazidime and tobramycin in zein nanoparticles coated with chitosan and to characterize and evaluate the antibacterial and antibiofilm activity against antibiotic-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae. Zein nanoparticles, synthesized using the nanoprecipitation method, were characterized by their particle size (Ø), polydispersity index (PDI), zeta potential (ζ), pH, and encapsulation efficiency (%EE). The chitosan coating provided stability, and physicochemical analyses revealed chemical interactions, efficient drug encapsulation, and thermal stability. The release kinetics demonstrated controlled release in simulated gastric and intestinal pH. The antibacterial activity, assessed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), indicated effectiveness against both pathogens. Antibiofilm assays, conducted using the crystal violet method, demonstrated the inhibition and eradication of biofilms. The chitosan-coated zein nanoparticles with CAZ and/or TOB exhibited Ø (315-335 nm), PDI (<0.2), ζ (+40 to +50 mV), pH (5), and %EE (>55%). Notably, the co-encapsulation formulation (CAZ-TOB-ZNP-CH) showed enhanced antibacterial and antibiofilm activities compared to the individual formulations. These findings suggest that the developed nanoparticles present a promising alternative for treating respiratory and intestinal infections caused by antibiotic-resistant and biofilm-producing P. aeruginosa and K. pneumoniae.

Olive oil nanoemulsion containing curcumin: antimicrobial agent against multidrug-resistant bacteria.

The present work aimed to develop, characterize, and evaluate the antibacterial and antibiofilm activity of two nanoemulsions (NEs) containing 500 µg/mL of curcumin from Curcuma longa (CUR). These NEs, produced with heating, contain olive oil (5%) and the surfactants tween 80 (5%) and span 80 (2.5%), water q.s. 100 mL, and were stable for 120 days. NE-2-CUR presented Ø of 165.40 ± 2.56 nm, PDI of 0.254, ζ of - 33.20 ± 1.35 mV, pH of 6.49, and Entrapment Drug Efficiency (EE) of 99%. The NE-4-CUR showed a Ø of 105.70 ± 4.13 nm, PDI of 0.459, ζ of - 32.10 ± 1.45 mV, pH of 6.40 and EE of 99.29%. Structural characterization was performed using DRX and FTIR, thermal characterization using DSC and TG, and morphological characterization using SEM, suggesting that there is no significant change in the CUR present in the NEs and that they remain stable. The MIC was performed by the broth microdilution method for nine gram-positive and gram-negative bacteria, as well as Klebsiella pneumoniae clinical isolates resistant to antibiotics and biofilm and efflux pump producers. The NEs mostly showed a bacteriostatic profile. The MIC varied between 125 and 250 µg/mL. The most sensitive bacteria were Staphylococcus aureus and Enterococcus faecalis, for which NE-2-CUR showed a MIC of 125 µg/mL. The NEs and ceftazidime (CAZ) interaction was also evaluated against the K. pneumoniae resistant clinical isolates using the Checkerboard method. NE-2-CUR and NE-4-CUR showed a synergistic or additive profile; there was a reduction in CAZ MICs between 256 times (K26-A2) and 2 times (K29-A2). Furthermore, the NEs inhibited these isolates biofilms formation. The NEs showed a MBIC ranging from 15.625 to 250 µg/mL. Thus, the NEs showed physicochemical characteristics suitable for future clinical trials, enhancing the CAZ antibacterial and antibiofilm activity, thus becoming a promising strategy for the treatment of bacterial infections caused by multidrug-resistant K. pneumoniae. KEY POINTS: • The NEs showed physicochemical characteristics suitable for future clinical trials. • The NEs showed a synergistic/additive profile, when associated with ceftazidime. • The NEs inhibited biofilm formation of clinical isolates.

Occurrence of blaNDM-7 and association with blaKPC-2, blaCTX-M15, aac, aph, mph(A), catB3 and virulence genes in a clinical isolate of Klebsiella pneumoniae with different plasmids in Brazil.

To characterize phenotypically and genotypically an isolate of multidrug-resistant (MDR) K. pneumoniae from a patient with septicemia in a hospital in Recife-PE, Brazil, resistance and virulence genes were investigated using PCR and sequencing the amplicons, and the plasmid DNA was also sequenced. The K74-A3 isolate was resistant to all ß-lactams, including carbapenems, as well as to aminoglycosides and quinolones. By conducting a PCR analysis and sequencing, the variants blaNDM-7 associated with blaKPC-2 and the cps, wabG, fim-H, mrkD and entB virulence genes were identified. The analysis of plasmid revealed the presence of blaCTX-M15, aac(3)-IVa, aph(3')-Ia, aph(4)-Ia, aac(6')ib-cr, mph(A) and catB3, and also the plasmids IncX3, IncFIB, IncQ1, ColRNAI and ColpVC. To our knowledge, this is the first report of the blaNDM-7 gene in Recife-PE and we suggest that this variant is located in IncX3. These results alert us to the risk of spreading an isolate with a vast genetic arsenal of resistance, in addition to which several plasmids are present that favor the horizontal transfer of these genes.

Virulence factors of Proteus mirabilis clinical isolates carrying blaKPC-2 and blaNDM-1 and first report blaOXA-10 in Brazil.

INTRODUCTION: Proteus mirabilis is one of the main pathogens that cause urinary tract infections. Therefore, the aim of this study was to analyze and compare the genetic profile of 36 clinical isolates of P. mirabilis that carry and do not carry the blaKPC and blaNDM gene with respect to virulence factors (mrpG, pmfA, ucaA, nrpG and pbtA) and antimicrobial resistance (blaVIM,blaIMP, blaSPM, blaGES,blaOXA-23-like, blaOXA-48-like, blaOXA-58-like and blaOXA-10-like). METHODS: The virulence and resistance genes were investigated by using PCR and sequencing. RESULTS: ERIC-PCR typing showed that the isolates showed multiclonal dissemination and high genetic variability. The gene that was most found blaOXA-10-like (n = 18), followed by blaKPC (n = 10) and blaNDM (n = 8). To our knowledge, this is the first report of blaOXA-10 in P. mirabilis in Brazil, as well as the first report of the occurrence of P. mirabilis co-carrying blaOXA-10/blaKPC and blaOXA-10/blaNDM. The blaNDM or blaKPC carrier isolates showed important virulence genes, such as ucaA (n = 8/44.4%), pbtA (n = 10/55.5%) and nrpG (n = 2/11.1%). However, in general, the non-carrier isolates of blaKPC and blaNDM showed a greater number of virulence genes when compared to the carrier group. CONCLUSION: Clinical isolates of P. mirabilis, in addition to being multi-drug resistant, presented efficient virulence factors that can establish infection outside the gastrointestinal tract.

High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla KPC and bla NDM from patients at a public hospital in Brazil.

INTRODUCTION: Antibiotic resistance in carbapenemase-producing Klebsiella pneumoniae is acquired and disseminated mainly by plasmids. Therefore, we aimed to investigate the occurrence of carbapenemase genes, analyze the genetic diversity by ERIC-PCR, and examine the most common plasmid incompatibility groups (Incs) in clinical isolates of K. pneumoniae from colonization and infection in patients from a hospital in Brazil. METHODS: Twenty-seven isolates of carbapenem-resistant K. pneumoniae were selected and screened for the presence of carbapenemase genes and Incs by PCR, followed by amplicon sequencing. RESULTS: The bla KPC and bla NDM genes were detected in 24 (88.8 %) and 16 (59.2 %) of the isolates, respectively. Thirteen isolates (48.1 %) were positive for both genes. The IncFIB (92.6 %) and IncQ (88.8 %) were the most frequent plasmids, followed by IncA/C, IncHI1B, and IncL/M, indicating that plasmid variability existed in these isolates. To our knowledge, this is the first report of IncHI1B in Brazil. We found eight isolates with clonal relationship distributed in different sectors of the hospital. CONCLUSIONS: The accumulation of resistance determinants, the variability of plasmid Incs, and the clonal dissemination detected in K. pneumoniae isolates demonstrate their potential for infection, colonization, and the dissemination of different resistance genes and plasmids.

Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil.

INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS: To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

In vitro activity of polymyxin B in combination with meropenem, amikacin and gentamicin against Klebsiella pneumoniae clinical isolates co-harbouring aminoglycoside-modifying enzymes, blaNDM-1 and blaKPC-2.

OBJECTIVES: Multidrug-resistant Klebsiella pneumoniae carrying blaNDM-1 and blaKPC-2 genes are a worldwide concern for which combination antimicrobial therapy may be the only viable option. The aim of this study was to investigate the in vitro activity of combinations of polymyxin B (PMB) with meropenem (MEM), amikacin (AMK) and gentamicin (GEN) at subinhibitory concentrations against two K. pneumoniae clinical isolates co-harbouring blaNDM-1, blaKPC-2 and aminoglycoside-modifying enzymes and resistant to PMB. METHODS: Synergy and bactericidal activity were evaluated by chequerboard and time-kill assays against two PMB-resistantK. pneumoniae clinical isolates carrying the blaNDM-1, blaKPC-2, aac(3)-IIa, aac(6')-Ib, aph(3')-VI and ant(2'')-Ia genes. Five combinations of PMB, MEM, AMK and GEN were evaluated. RESULTS: The PMB/MEM and PMB/AMK combinations proved to be the best options against isolate K7R2, mainly because they demonstrated bactericidal activity when using subinhibitory concentrations of these antimicrobials. However, none of the studied combinations was bactericidal against isolate K11R2. CONCLUSION: The combinations used in this study showed synergy against NDM-and KPC-producing isolates but, given their bactericidal activity, the combinations of PMB/MEM and PMB/AMK were the most active against one isolate. It can also be concluded that the antimicrobials to which the bacteria were resistant could form part of combination therapy.

High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla KPC and bla NDM from patients at a public hospital in Brazil

Abstract INTRODUCTION Antibiotic resistance in carbapenemase-producing Klebsiella pneumoniae is acquired and disseminated mainly by plasmids. Therefore, we aimed to investigate the occurrence of carbapenemase genes, analyze the genetic diversity by ERIC-PCR, and examine the most common plasmid incompatibility groups (Incs) in clinical isolates of K. pneumoniae from colonization and infection in patients from a hospital in Brazil. METHODS Twenty-seven isolates of carbapenem-resistant K. pneumoniae were selected and screened for the presence of carbapenemase genes and Incs by PCR, followed by amplicon sequencing. RESULTS The bla KPC and bla NDM genes were detected in 24 (88.8 %) and 16 (59.2 %) of the isolates, respectively. Thirteen isolates (48.1 %) were positive for both genes. The IncFIB (92.6 %) and IncQ (88.8 %) were the most frequent plasmids, followed by IncA/C, IncHI1B, and IncL/M, indicating that plasmid variability existed in these isolates. To our knowledge, this is the first report of IncHI1B in Brazil. We found eight isolates with clonal relationship distributed in different sectors of the hospital. CONCLUSIONS The accumulation of resistance determinants, the variability of plasmid Incs, and the clonal dissemination detected in K. pneumoniae isolates demonstrate their potential for infection, colonization, and the dissemination of different resistance genes and plasmids.

Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

Emergence of bla NDM-1 associated with the aac(6')-Ib-cr, acrB, cps, and mrkD genes in a clinical isolate of multi-drug resistant Klebsiella pneumoniae from Recife-PE, Brazil.

INTRODUCTION: The emergence of New Delhi metallo-ß-lactamase (NDM) is concernig because it reduces the antibiotic therapy options for bacterial infections. METHODS: Resistant and virulent genes from an isolate of Klebsiella pneumoniae derived from a patient with sepsis in a hospital in Recife-PE, Brazil, were investigated using PCR and DNA sequencing. RESULTS: bla NDM-1, aac(6')-Ib-cr and acrB resistance genes, and cps and mrkD virulence genes were detected. CONCLUSIONS: To our knowledge, this is the first report on bla NDM-1 in Recife-PE. This detection alerts researchers to the need to control the spread of bla NDM-1 resistance gene by this bacterium in Brazil.

Emergence of bla NDM-1 associated with the aac(6')-Ib-cr, acrB, cps, and mrkD genes in a clinical isolate of multi-drug resistant Klebsiella pneumoniae from Recife-PE, Brazil

Abstract INTRODUCTION: The emergence of New Delhi metallo-β-lactamase (NDM) is concernig because it reduces the antibiotic therapy options for bacterial infections. METHODS: Resistant and virulent genes from an isolate of Klebsiella pneumoniae derived from a patient with sepsis in a hospital in Recife-PE, Brazil, were investigated using PCR and DNA sequencing. RESULTS: bla NDM-1, aac(6')-Ib-cr and acrB resistance genes, and cps and mrkD virulence genes were detected. CONCLUSIONS To our knowledge, this is the first report on bla NDM-1 in Recife-PE. This detection alerts researchers to the need to control the spread of bla NDM-1 resistance gene by this bacterium in Brazil.

Occurrence of qnrB1 and qnrB12 genes, mutation in gyrA and ramR, and expression of efflux pumps in isolates of Klebsiella pneumoniae carriers of blaKPC-2.

PURPOSE: The occurrence of quinolone-resistance genes (qnrA, qnrB and qnrS), the presence of mutations in gyrA, gyrB and parC, as well as the expression of efflux pumps (acrB and acrF) and mutations in the gene ramR. METHODOLOGY: Were investigated in 30 blaKPC-2-positive isolates of Klebsiella pneumoniae taken from infection and colonization in hospital patients from Recife-PE, Brazil. The detection of the qnr, acrB and acrF genes and analysis of the mutations in ramR and the quinolone-resistance-determining regions of gyrA, gyrB and parC were performed by PCR followed by DNA sequencing. RESULTS: Among the isolates analysed, 73.3 % (n=22) presented the qnrB gene. For the DNA sequencing, six isolates (K3-A2, K12-A2, K25-A2, K27-A2, K19-A2 and K3-C2) were selected and the qnrB1 and qnrB12 variants were detected. This is the first ever report, to the best of our knowledge, of the presence of qnrB12 in K. pneumoniae. This is also the first report, to the best of our knowledge, of the presence of qnrB1 or qnrB12 with blaKPC-2 in K. pneumoniae in Brazil. Mutations were observed in gyrA S83 and in ramR. All isolates presented genes for the acrB and acrF efflux pumps and the reverse transcription PCR performed showed that the pumps were being expressed. CONCLUSION: KPC-2-positive isolates colonizing patients, which also showed qnrB, mutation in gyrA and efflux pumps, may be important reservoirs for disseminating these resistance mechanisms in the hospital environment.

Ultrastructural changes caused by polymyxin B and meropenem in multiresistant Klebsiella pneumoniae carrying blaKPC-2 gene.

The ultrastructural alterations caused by polymyxin B and meropenem and by the association between polymyxin B and meropenem were investigated in two multiresistant isolates of Klebsiella pneumoniae (K3-A2 and K12-A2) carriers of blaKPC-2, coming from infection and colonization in patients in a public hospital in Recife, Brazil. The ultrastructural changes were detected by transmission electron microscopy and scanning. The susceptibility of the isolates to antimicrobials was tested by the disc diffusion method and microdilution in broth. The analysis by electron microscopy showed that the isolates presented morphological and ultrastructural cellular changes when subjected to a clinically relevant concentration of antimicrobials alone or in combination. When subjected to meropenem, they presented retraction of the cytoplasmic material, rupture of the cell wall and extravasation of the cytoplasmic content. When submitted to polymyxin B, the isolates showed condensation of the ribosomes, DNA clotting, cell wall thickening and the presence of membrane compartment. When subjected to polymyxin B and meropenem in combination, the isolates showed a higher intensity of the ultrastructural changes visualized. This is the first report of the ultrastructural changes caused by polymyxin B and meropenem in multiresistant isolates of K. pneumoniae carriers of the blaKPC-2 gene. It should be noted that even when the K. pneumoniae isolates were multiresistant carriers of the blaKPC-2 gene, they underwent important structural change owing to the action of polymyxin B and meropenem.