Cytocompatibility of poly(p-dioxanone)/ poly(hydroxybutic) (PPD/PHB) blends to cartilage tissue engineering

In order of seek strategies to improve the interaction between bioreabsorbable polymer materials and cellular growth, this work aimed at evaluating in vitro the influence of PPD/PHB blends on cell adhesion and fibrochondrocytes growth. Fibrochondrocytes cells were obtained by primary extraction from enzymatic digestion methods. The PPD/PHB blends were prepared by casting with 100/0, 60/40 and 50/50 compositions, and were characterized by scanning electron microscopy (SEM). After 6, 48, 120 and 168 hours in culture, ultrastructural observations showed changes in cell morphology, suggesting that the fibrochondrocytes can respond to substrate modifications, changing their phenotypic profile. The MTT analyses showed that the blends did not present cytotoxicity and allowed fibrochondrocytes adhesion and proliferation on the membranes in all compositions. The colorimetric Sirius Red test revealed the capability of extracellular matrix synthesis on the blends, from which one can conclude that the PPD/PHB blends are not cytotoxic and can be indicated for cell culture.Buscando estratégias que repercutam na melhoria da interação entre materiais poliméricos biorreabsorvíveis e o crescimento celular, o presente estudo in vitro teve como objetivo estudar a influência de blendas de PPD/PHB na adesão celular e crescimento de fibrocondrócitos obtidos a partir de cultura primária. As blendas de PPD/PHB foram preparadas pelo método de evaporação de solvente nas composições 100/0, 60/40 e 50/50 e caracterizadas por microscopia eletrônica de varredura (MEV). Observações ultra-estruturais mostraram alterações na morfologia celular, sugerindo que os fibrocondrócitos podem responder a alterações no substrato alterando seu perfil fenotípico. As análises com MTT demonstraram que as blendas não apresentaram citotoxicidade e permitiram a adesão e proliferação dos fibrocondrócitos sobre os substratos em todas as suas composições. O ensaio colorimétrico com Sirius Red evidenciou a capacidade de manutenção da síntese de matriz extracelular colágena sobre as amostras, concluindo-se que as blendas de PPD/PHB podem ser indicadas para o cultivo celular.383388Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

PLLA/Triethyl citrate membrane as an alternative for the treatment of skin wounds

Bioresorbable polymers can be applied as membranes to sustain and guide cell growth through the regeneration process. This study evaluated poly(acid lactide), PLLA, membranes with addition of 10% triethyl citrate as skin wound healing in Wistar rats. Initially a 2cm² skin wound was exercised of the back of 24 animals. The animals were divided into two groups: treated, whose the polymer membrane was implanted, and control, in which the wound was kept exposed. The results obtained after 1, 3, 7 and 15 days showed an inflammatory response more satisfactory in the implanted wounds, with early repair and collagen more organized when compared to exposed wounds. In addition to, the protected areas showed no irritant inflammatory response which could be attributed to the membrane. Thus, we conclude that the PLLA/Triethyl citrate membrane has effectively protected the wounds, allowing the repair and presenting itself as a promising skin dressing.Polímeros sintéticos biorreabsorvíveis podem ser utilizados sob a forma de membranas para sustentar e guiar o crescimento celular, através do processo de reparação tecidual. Este trabalho avaliou membranas de poli(ácido lático), PLLA, com adição de 10% de trietil-citrato usadas como curativos de feridas cutâneas agudas em ratos Wistar. Inicialmente uma ferida de 2cm² foi provocada na região dorsal de 24 animais. Estes foram divididos em 2 grupos: tratamento, nos quais as feridas foram recobertas pela membrana polimérica e controle, com feridas permanecendo cruentas. Os resultados obtidos em 1, 3, 7 e 15 dias mostraram uma resposta inflamatória mais satisfatória nas feridas protegidas pelas membranas, com reparação precoce e colágeno mais organizado quando comparadas com as áreas incialmente mantidas sem proteção. Além do que, as áreas protegidas pelas membranas não mostraram alterações inflamatórias irritativas que pudessem ser imputadas ao uso da membrana polimérica. Diante disso, conclui-se que a membrana de PLLA/Trietil-citrato protegeu efetivamente as feridas, permitindo o processo de reparação e mostrando-se promissora como curativo cutâneo.798806Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy

Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD50 and LD90) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates

A novel IncA/C1 group conjugative plasmid, encoding VIM-1 metallo-beta-lactamase, mediates the acquisition of carbapenem resistance in ST104 Klebsiella pneumoniae isolates from neonates in the intensive care unit of V. Monaldi Hospital in Naples

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim-sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2'-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples

WHO Critical Priority Escherichia coli as One Health Challenge for a Post-Pandemic Scenario: Genomic Surveillance and Analysis of Current Trends in Brazil.

The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum β-lactamase (ESBL)-positive strains carrying a wide diversity of variants, and the growing number of colistin-resistant isolates carrying -type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored and genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts

Citocompatibilidade de blendas de poli(p-dioxanona)/ poli(hidroxi butirato) (PPD/PHB) para aplicações em engenharia de tecido cartilaginoso Cytocompatibility of poly(p-dioxanone)/ poly(hydroxybutic) (PPD/PHB) blends to cartilage tissue engineering

Buscando estratégias que repercutam na melhoria da interação entre materiais poliméricos biorreabsorvíveis e o crescimento celular, o presente estudo in vitro teve como objetivo estudar a influência de blendas de PPD/PHB na adesão celular e crescimento de fibrocondrócitos obtidos a partir de cultura primária. As blendas de PPD/PHB foram preparadas pelo método de evaporação de solvente nas composições 100/0, 60/40 e 50/50 e caracterizadas por microscopia eletrônica de varredura (MEV). Observações ultra-estruturais mostraram alterações na morfologia celular, sugerindo que os fibrocondrócitos podem responder a alterações no substrato alterando seu perfil fenotípico. As análises com MTT demonstraram que as blendas não apresentaram citotoxicidade e permitiram a adesão e proliferação dos fibrocondrócitos sobre os substratos em todas as suas composições. O ensaio colorimétrico com Sirius Red evidenciou a capacidade de manutenção da síntese de matriz extracelular colágena sobre as amostras, concluindo-se que as blendas de PPD/PHB podem ser indicadas para o cultivo celular.In order of seek strategies to improve the interaction between bioreabsorbable polymer materials and cellular growth, this work aimed at evaluating in vitro the influence of PPD/PHB blends on cell adhesion and fibrochondrocytes growth. Fibrochondrocytes cells were obtained by primary extraction from enzymatic digestion methods. The PPD/PHB blends were prepared by casting with 100/0, 60/40 and 50/50 compositions, and were characterized by scanning electron microscopy (SEM). After 6, 48, 120 and 168 hours in culture, ultrastructural observations showed changes in cell morphology, suggesting that the fibrochondrocytes can respond to substrate modifications, changing their phenotypic profile. The MTT analyses showed that the blends did not present cytotoxicity and allowed fibrochondrocytes adhesion and proliferation on the membranes in all compositions. The colorimetric Sirius Red test revealed the capability of extracellular matrix synthesis on the blends, from which one can conclude that the PPD/PHB blends are not cytotoxic and can be indicated for cell culture

Image_3_Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy.PDF

<p>Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD₅₀ and LD₉₀) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.</p