ABSTRACT
Pseudomonas aeruginosa es uno de los patógenos oportunistas más importantes, causante de infecciones, con altos índices de morbilidad y mortalidad. Los carbapenemes son antibióticos que poseen un amplio espectro de actividad y son altamente potentes, lo cual hacen que sean imprescindibles en el tratamiento empírico. P. aeruginosa presenta diversos mecanismos de resistencia, entre ellos las carbapenemasas tipo metalo-β-lactamasas (MBLs). Debido a los numerosos reportes de bacterias productoras de MBLs, es importante la aplicación de test simples, prácticos y de bajo costo, como pruebas de rutina, para que se pueda identificar a las bacterias productoras de MBLs de forma rápida. El objetivo de este trabajo es determinar fenotípicamente la presencia de carbapenemasas en aislamientos de P. aeruginosa. Estudio prospectivo, descriptivo de corte transversal realizado en aislamientos de P. aeruginosa de pacientes que acudieron al Hospital de Clínicas - San Lorenzo en el periodo de febrero a julio de 2013. Se estudiaron 232 aislamientos de P. aeruginosa, a aquellos con sospecha de carbapenemasas se les aplicó dos métodos de detección fenotípica, discos de EDTA y discos con ácido dipicolinico - Meropenem (DPA-ME). De estos aislamientos, 30 dieron sinergia con la técnica de EDTA y 18 aislamientos positivos con los discos de DPA. A través de los métodos fenotípicos aplicados se pudo comprobar la presencia de cepas productoras de carbapenemasas tipo MBL en una frecuencia de 7,8%. Los tests de combinación de disco podrían ser útiles en la práctica diaria para proporcionar una detección rápida y fiable de MBL carbapenemasas en los aislados de P. aeruginosa cuando las pruebas moleculares no están disponibles.
P. aeruginosa is one of the most important opportunistic pathogens that cause infections with high morbidity and mortality. Carbapenems are antibiotics with a broad spectrum of activity and highly powerful, which makes them indispensable in the empirical treatment. P. aeruginosa has various mechanisms of resistance, including metallobetalactamase (MBL) type carbapenemases. Due to increasing numbers of MBL producing bacteria, it is important to apply simple tests that are practical and inexpensiveas routine protocol in order to rapidly identify MBL producing bacteria. The objective ofthis study was to determine phenotypically the presence of carbapenemases in P aeruginosa isolates. A descriptive cross- sectional study was performed in isolates of P.aeruginosa from patients attending the Hospital de Clinicas - San Lorenzo from February to July, 2013. Two hundred thirty two isolates of P. aeruginosa were studied. Those isolates suspicious of having Carbapenemases were subjected to two phenotypic detection methods: discs of EDTA and discs of dipicolinic acid Meropenem (DPA-ME). Of these isolates, 30 were synergistic with the technique of EDTA and 18 positive with DPA discs.The presence of strains producing MBL type carbapenemases was determined throughthese phenotypic methods yielding a frequency of 7.8%. The disc combination tests maybe very useful in the daily practice to provide fast and reliable detection of MBL carbapenemases in P. aeruginosa isolates, where molecular biology tests are not available.
Subject(s)
Humans , Pseudomonas Infections , Pseudomonas aeruginosaABSTRACT
BACKGROUND: Since metallo-beta-lactamase (MBL)-producing isolates can hydrolyze carbapenem and also easily transfer the resistance genes to other bacteria, a rapid and accurate detection of MBL has become very important. We evaluated the utility of Mueller Hinton agar (MHA) biplate containing dipicolinic acid (DPA) as a screening method to detect IMP-1 and VIM-2 type MBL-producing isolates. METHODS: Based on our preliminary tests using various concentrations of DPA, 200 and 300 microg/mL concentration of DPA were chosen for further study. Bacterial lawns were grown on MHA biplate, one half of which contained DPA while the other did not. The inhibition zone around the imipenem (IPM) disk on both sides of this plate was compared. The stability of DPA in the stored DPA-MHA biplate was also evaluated during three months using two MBL- and one non-MBL-producing isolates. RESULTS: When the criterion of a > or =7 mm increase of inhibition zone around the IPM disk on the MHA containing DPA compared to MHA without DPA was used, the sensitivities and specificities were 94.7% and 97.6% for 200 microg/mL DPA-MHA biplate, and 98.2% and 97.6% for 300 microg/mL DPA-MHA biplate, respectively. The activity of the DPA in this biplate was stable for three months. CONCLUSIONS: Assays using DPA 300-MHA biplate were highly sensitive and specific for the detection of IMP-1 and VIM-2 type MBL-producing bacteria. In addition, it is easy to perform; so, it may be useful to apply this method for detection of IMP-1 and VIM-2 type MBL in clinical laboratories.
Subject(s)
Agar , Anti-Bacterial Agents/pharmacology , Bacteriological Techniques , Chelating Agents/chemistry , Drug Resistance, Bacterial , Gram-Negative Bacteria/drug effects , Imipenem/pharmacology , Picolinic Acids/chemistry , Reagent Kits, Diagnostic , Sensitivity and Specificity , beta-Lactamases/analysisABSTRACT
Rapidly metabolizable compounds such as glucose or glycerol were not utilized by Bacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores of Bacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism.
ABSTRACT
Dipicolinic acid synthesis in Penicillium citreoviride strain 3114 was inhibited by Ca2+ ions, but not by Ba2+, Cu2+or Fe2+. Among the metals tested, only Zn2+ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca2+ or Zn2+ . Α mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca2+ complex showed cross-resistance to inhibition by dipicolinic acid: Zn2+. Both 3114 and 27133-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca2+, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca2+ did not inhibit the de novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca2+ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca2+ complex, mutant differed from the parent in three other aspects viz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca2+ ions in the medium and (iii) Mg2+ requirement for the mutant increased three fold. Higher requirement of the Mg2+ could be partially relieved by Ca2+ during secondary metabolism. The results support the inference that de novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca2+ complex.
ABSTRACT
Synthesis of dipicolinic acid in Penicillium citreoviride showed typical kinetics of a secondary metabolite. Its synthesis resumed during idiophase and continued through stationary phase of growth. Total duration of synthesis was 100 h at the end of which its synthesis was arrested. Production of dipicolinic acid by the cells was subject to catabolite repression by glucose and was not subject to end product inhibition by exogenously added dipicolinic acid. Unlike the bacteria, dipicolinic acid synthesis in this mold was highly sensitive to inhibition by calcium ions in the growth medium. Calcium promoted sporulation but dipicolinic acid was not found to be present in detectable amounts in mold spores. Addition of dipicolinic acid and Ca2+ completely inhibited its de novo synthesis, an effect not observed when calcium was replaced by Mg2+ When the mold was grown in the presence of calcium alone, its inhibitory effects on de novo synthesis of dipicolinic acid were expressed only after some of this metabolite was first synthesised by the producer cells suggesting that the active feedback inhibitor is probably a Ca: dipicolinic acid complex. It is suggested that over-production of this metabolite is very important to the mold in increasing its survival potential in nature by retrieving the essential minerals from the environment through ligand: metal complex at a time when cells are in the process of dying, so that a proper mineral balance is maintained within the cells.
ABSTRACT
Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn2+ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn2+ However, the levels of the inhibitor decreased considerably (75-85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn2+ and Ca2+ as determined by Scatchard plot analysis.