RESUMO
La biopelícula como un mecanismo de virulencia en Staphylococcus involucrada en infecciones intrahospitalarias es regulada por un represor negativo icaR, responsable de la transcripción completa del operón icaADBC. La búsqueda de dominios funcionales por modulación computacional de icaR permitió hallar las secuencias peptídicas con actividad biológica análoga a la proteína icaR. Mediante biología computacional se diseñaron péptidos empleando el programa de predicción AntiBP (http://www.imtech.res.in/raghava/antibp/); la síntesis química se hizo por Nα-Fmoc y se caracterizaron y purificaron tres moléculas por RP-HPLC y MALDI-TOF. Se evaluó su seguridad biológica mediante ensayo de actividad citotóxica realizada sobre macrófagos murinos de la línea J774 y la actividad hemolítica se determinó mediante el uso de glóbulos rojos. Los tres péptidos caracterizados IR1, IR2 e IR3, presentaron estructura secundaria predominantemente alfa helicoidal, alto grado de pureza y alto score antimicrobiano; además, mostraron baja toxicidad, evidenciada por la actividad citotóxica y hemolítica en las concentraciones ensayadas y en comparación con los controles usados, que permitiría su potencial uso como moléculas candidatas o principios activos con actividad análoga al represor nativo icaR, frente a la biopelícula de los Staphylococcus sp.
Staphylococcus sp. biofilm, formed as a mechanism of virulence that is involved in hospital acquired infections, is regulated by a negative repressor icaR, which is responsible for the full transcription of the operon icaADBC. This study, through functional commands by computational modulation of icaR, allowed to find peptide sequences with similar biological activity to the icaR protein. Peptides were designed by means of computational biology using the prediction program AntiBP (http://www.imtech.res.in/raghava/antibp/). The chemical synthesis of peptides was performed by Nα-Fmoc. The purification and characterization of three molecules were carried out using RP-HPLC and MALDI-TOF. Biological safety of peptides was evaluated by tests of cytotoxic activity on murine macrophage cells line J774, and their hemolytic activity was determined by using red cells. The three characterized peptides IR1, IR2 and IR3 presented a predominantly secondary alpha helical structure with a high degree of purity and high antimicrobial scores. In addition, the peptides exhibited low toxicity, proved by their low cytotoxic and hemolytic activity in the tested concentrations and in comparison to the standards used. These results allow the potential use of these peptides as candidate molecules or active principles with similar activity to the native repressor icaR against the Staphylococcus biofilm.
O biofilme formado como um mecanismo de virulência em Staphylococcus sp., que está envolvido com infecções intra-hospitalares, é regulado por um repressor negativo icaR, o qual é responsável pela plena transcrição do operão icaADBC. Por tanto, o presente estudo, avaliando a segurança biológica de moléculas, concebeu peptídeos antibiofilme semelhantes ao repressor icaR. Por meio da biologia computacional foram concebidos peptídeos usando o programa de predição AntiBP (http://www.imtech.res.in/raghava/antibp/) para identificar as sequências com uma atividade biologicamente similar à da proteína icaR. A Síntese química dos peptídeos se fez pelo Nα-Fmoc e foram caracterizadas e purificadas três moléculas por RP-HPLC e MALDI-TOF. O ensaio de atividade citotóxica foi realizado nos macrófagos murinos da linha J774 e a atividade hemolítica foi determinada por meio do uso de glóbulos vermelhos. Foram caraterizados três peptídeos IR1, IR2 e IR3, além de mostrarem uma estrutura secundaria predominantemente alfa helicoidal, com alto grau de pureza, alto score antimicrobiano e baixa toxicidade, estes podem ser postulados como moléculas candidatas ou princípios ativos com uma atividade similar ao repressor nativo icaR frente ao biofilme dos Staphylococcus sp.
RESUMO
OBJECTIVES: Investigating the prevalence and sensitivity of germs isolated from newborn in a referral hospital in Bogotá. Suggesting an empirical antibiotic treatment for neonatal infection. METHODS: Cultures taken between February and December 2002 were analysed. Blood cultures were processed using BacT/ALERT (Durham, NC), urine cultures by UROCULT (Bio-Bacter) and catheter tips in thioglycollate. BBL CRYSTAL identification system (BD, Sparks, MD) was used for identifying germs. Antibiotic sensitivity was determined by disk diffusion. RESULTS: There were 1,097 positive aerobic and facultative aerobic germ cultures; 64.3% were Gram-positive, 30.6% Gram-negative and 4.9% were yeasts. Gram-positive germs consisted of coagulase-negative staphylococci (64.2%), enterococcus (13.8%) and coagulase-positive staphylococci (13.3%). The most frequent Gram-negatives were Klebsiella (45.2%), Eschericha coli (30.9%) and Serratia (10.1%). Staphylococcus epidermidis accounted for 64% of the coagulase-negative staphylococci. S. epidermidis susceptibility to vancomycin was 100%. Coagulase-negative staphylococci susceptibility to rifampin and amikacin was 59% and 67.4% (respectively). Coagulase-negative staphylococci resistance to beta-lactams was 86.4% (95% CI: 82.3-89.9). Coagulase-positive staphylococci sensitivity to vancomycin was 100%. Gram-negative susceptibility to imipenem was 98.1% (95% CI: 89.9-99.9), 78.1% to gentamicin (95% CI: 64.9-88.2) and 46.6% to amikacin (95% CI: 28.3-65.7). CONCLUSIONS: There was high coagulase-negative staphylococci prevalence in neonatal infection (particularly S. epidermidis). All S. epidermidis and coagulase-positive staphylococci were sensitive to vancomycin. There was increasing coagulase-negative staphylococci and Gram-negative resistance to oxacillin and amikacin, respectively.
