Actividad inhibitoria de dihidroxifenil propenona sobre betalactamasas de Enterobacter cloacae: estudio preliminar en el desarrollo de fármacos para enfrentar la resistencia bacteriana / Inhibitory activity of dihydroxy-phenyl-propenone on betalactamase of Enterobacter cloacae : Preliminary study for drug development to overcome bacterial resistance

Introducción . Los microorganismos patógenos como Enterobacter cloacae producen betalactamasas que les confieren resistencia frente a los antibióticos betalactámicos; se ha identificado, además, la actividad limitada de los inhibidores enzimáticos, de modo que la única posibilidad de enfrentar la resistencia es el diseño de nuevos fármacos y su uso racional. Objetivo. Evaluar el efecto de la chalcona dihidroxifenil propenona sobre un aislamiento clínico de E. cloacae y sobre la betalactamasa aislada a partir de este microorganismo resistente como un aporte en la búsqueda de compuestos inhibidores de las betalactamasas. Materiales y métodos. Se sintetizó la chalcona dihidroxifenil propenona y se evaluó su efecto sobre el aislamiento clínico de E. cloacae para determinar la concentración inhibitoria mínima mediante el método de microdilución en caldo y con la betalactamasa purificada mediante cromatografía de afinidad se realizaron estudios espectrofotométricos de cinética enzimática. Resultados. La concentración inhibitoria mínima de la dihidroxifenil propenona sobre E. cloacae fue de 35 µg/ml; el porcentaje de recuperación de la betalactamasa a partir del microorganismo fue de 31,75 %; en el estudio cinético se evidenció actividad inhibitoria de acuerdo con los parámetros cinéticos de V max =1,7 x 10 -3 µM/minuto y K M´ =2330 µM. Conclusión. La chalcona dihidroxifenil propenona ejerce su actividad inhibitoria por medio de la interacción con la betalactamasa y, de esta manera, protege la integridad estructural de los antibióticos betalactámicos; dicho efecto sinérgico la convierte en un compuesto promisorio en la búsqueda de alternativas para enfrentar la resistencia bacteriana.

Introduction: Enterobacter cloacae is a pathogenic microorganism with the ability to produce betalactamase enzymes, which makes them resistant to betalactamic antibiotics. Additionally, the limited activity of enzymatic inhibitors has been identified, and, therefore, the design of new drugs and the promotion of their rational use are the only possibilities to overcome this problem. Objective: The aim of this research was to evaluate the effect of dihydroxy-phenyl-propenone on a clinical isolate of E. cloacae , as well as its activity on a betalactamase isolated from this resistant microorganism in order to contribute to the search for new betalactamase inhibitors. Materials and methods: Dihydroxy-phenyl-propenone chalcone was synthesized and evaluated on a clinical isolate of E. cloacae to determine the minimum inhibitory concentration by broth microdilution; once the betalactamase enzyme was purified by affinity chromatography, a spectrophotometric analysis was done to evaluate its kinetic activity. Results: The minimum inhibitory concentration value of dihydroxy-phenyl-propenone on E. cloacae was 35 µg/ml; the recovery percentage of the betalactamase from the microorganism was 31.75% and the kinetic parameters were V max =1.7 x 10 -3 µM/min and K M = 2330 µM, which show an important inhibitory activity. Conclusion: Dihydroxy-phenyl-propenone has shown inhibitory activity on betalactamase enzymes and the ability to protect the chemical integrity of betalactamic antibiotics; this synergistic effect turns it into a promising compound in the search for new alternatives to overcome bacterial resistance.

Study of beta lactamase activity of Staphylococcus aureus isolated from healthy nasal carriers and hospital isolates.

Staphylococcus aureus (n=84) isolated from the nostrils of a healthy population from Kathmandu and from the infectious cases (n=100) from Tribhuvan University Teaching Hospital, Kathmandu, Nepal were tested from May 1996 to March 1997 in Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal by microbiological and chemical methods to find out their beta lactamase activity. Among the healthy population, in domiciliary conditions 21.4% of the isolates were found beta lactamase producers. The occurrence of beta lactamase producing S. aureus was greater among female (27.0%) than among male (17.0%), however it was not significant (X2 = 1.2309, P > 0.05). The occurrence of the same was observed high among 40 and above age groups (66.7%) and 0-9 age group (60.0%), however no association with any particular age group was observed (X2 = 16.8674, P > 0.05). The b lactamase activity of S. aureus hospital inpatients isolates was 75.0% showing high occurrence of b lactamase activity in hospital isolates compared to S. aureus isolates from healthy carriers (X2 = 52.4113, P < 0.001). No association of beta lactamase positive hospital isolates with gender (X2 = 0.2158, P > 0.05) and age group (X2 = 1.5522, P > 0.05) was observed. This study shows that the prevalence of beta lactamase positive S. aureus was greater in hospital cases than in nasal carriers in domiciliary condition indicating the requisition of further study in this field.

ELISA using enzyme penicillinase for the detection of IgG antibodies to Trypanosoma evansi in sera of experimentally infected rabbits.

An Enzyme Linked Immunosorbent Assay (ELISA) using penicillinase as an enzyme marker has been developed for the detection of IgG antibodies in the sera of Trypanosoma evansi infected rabbits. Anti-T. evansi IgG antibodies were detected on 10th day after the infection and thereafter antibody titre rose progressively for 18 days. The developed assay is simple (end result assessed visually) and reproducible (4.4 and 14.0 percentage of intra and inter coefficient of variation respectively).

Activity and stability of Bacillus cereus penicillinase entrapped in aerosol OT reverse micelles.

The kinetics of enzyme catalyzed hydrolysis of penicillin G and stability of the enzyme alpha-penicillinase, entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, such as, water pool size (related to Wo), pH and temperature, were optimized for maximum activity of penicillinase in water/aerosol OT/isooctane reverse micelles. The enzyme showed maximum activity of Wo - 14 and pH, 7.0. At any temperature the enzyme was to be more active in reverse micelles than in aqueous solution. At optimum conditions of Wo, pH and temperature the enzyme was 100% more active in reverse micelles than its maximum activity in aqueous solution. In both the systems, the activity starts falling at and above 25 degrees C. CD Spectral studies showed that the enzyme in reverse micelles possesses more helical structure than it has in aqueous solution and at the optimum conditions in which it showed maximum activity, the alpha-helicity was also maximum. The enzyme was very stable in reverse micelles at and above room temperature compared to the same in aqueous solution.

Correlation between beta-lactamase production and MIC values against penicillin with coagulase negative staphylococci.

Two hundred strains of coagulase negative staphylococci (CNS) isolated from various clinical specimens (116) and healthy hospital personnel (84) were investigated for the production of beta-lactamases by means of three iodometric techniques and correlated with the minimum inhibitory concentration (MIC) values of penicillin-G by agar dilution technique and disc diffusion technique. One hundred and fifty (75.0%) of the 200 strains tested produced beta-lactamases. Seventy two per cent of the CNS were found to be beta-lactamase positive by the starch paper technique which was the most sensitive one in our study. The MIC values of penicillin against CNS ranged from less than or equal to 1.25 to greater than or equal to 2000 units. The present study indicated the higher prevalence of beta-lactamase producers with increased penicillin resistance among CNS strains isolated from healthy carriers and hospitalised patients.

Penicillinase-producing Neisseria gonorrhoeae in Dhaka, Bangladesh.

Among the 52 strains of Neisseria gonorrhoeae isolated from cases of acute urethritis attending the dermatovenerology department of different hospitals of Dhaka city, 5(9.6%) strains were identified as penicillinase-producing (PPNG) and had minimum inhibitory concentration (MIC) of penicillin 4.0 micrograms/ml or more. Of the rest 47 (90.4%) non-penicillinase-producing strains (non-PPNG), 18(38.3%) were fully sensitive to penicillin (MIC = 0.06 micrograms/ml or less), 25(53.2%) had diminished sensitivity (MIC = 0.125 to 0.5 micrograms/ml) and 4(8.5%) were resistant to penicillin (3 had MIC 1 micrograms/ml and 1 had MIC 2 micrograms/ml).

A penicillinase test incorporated in the rapid carbohydrate fermentation method for rapid detection of beta-lactamase producing Neisseria gonorrhoeae.

A modification of the acidometric (phenol red) test for penicillinase producing N. gonorrhoeae was incorporated into the rapid fermentation method for rapid screening and identification of PPNG strains. Two hundred and twenty-four non-penicillinase-producing N. gonorrhoeae, 55 penicillinase-producing N. gonorrhoeae, 87 N. meningitidis and 89 N. lactamica were included in this study. Results of the modified test were comparable with the iodometric and penicillin disk diffusion susceptibility and were obtainable within 1 to 5 minutes.