Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against ...multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and β-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin-chloramphenicol for infections caused by colistin-resistant E. cloacae.
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•Evaluation of 35 Aspergillus strains isolated from Brazil for lovastatin and itaconic acid production.•Molecular identification enabled to separate the 35 Aspergillus terreus strains ...in three monophyletic groups.•Lovastatin production stands out in the monophyletic group A. terreus B.•There is no specific monophyletic group for itaconic acid producing strains.•Identification of seven Brazilian strains with different levels of lovastatin and itaconic acid production.•Lovastatin genomic data was corroborated by metabolite detection using LC-MS.
Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.
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Previous issue date: 2014-01-01
The identification of filamentous fungiis traditionally based on morphological and biochemical criteria which are time demanding and for which results can be inaccurate. This fact motivated the evaluation of matrix-assisted laser desorption ionization ??? time of flight (MALDI-TOF) mass spectrometry for the identification of 36 brazilian isolates of Aspergillus terreus. The species is important worldwide due to its ability to produce lovastatin, a drug extremely successful in decreasing plasmatic cholesterol and consequently decreasing the risk of cardiovascular disease, main cause of death worldwide. Prior to MS analysis, the sequencing of internal transcribed spacer (ITS) allowed the identity confirmation of 94% of samples when compared GenBank database. First of all with the aim to compare the molecular biological technique with identification for MALDI-TOF was evaluated the influence of important factors in the quality of mass spectrums and then selected those more suitable to the identification of the specie. This enabled to characterize the proteic profile 30 of the 36 isolates. Nevertheless only 20% of brazilian isolates were identified as A. terreus when compared at the almost 4000 organisms of Biotyper database which may be attributed to the low representation of the specie. The results show that there are strong evidences that the technique is suitable which corroborates with this is: the similar pattern in the dendograms generated for each methodology, the ability to distinguish of the isolates comparatively higher by MALDI-TOF and the proven effectiveness of the technique evaluated by dark tests from the construction of a personal library. For these reasons the use of MALDI-TOF in the identification of isolates of A. terreus in replacing the conventional techniques is promising nevertheless requires optimizations regarding standardizing the processing of samples and inclusion of more reference spectra in the specie in database.
A identifica???o de fungos filamentosos ?? tradicionalmente baseada em crit??rios morfol??gicos e bioqu??micos que demandam tempo e cujos resultados podem ser imprecisos. Isso motivou a avalia???o do uso da t??cnica alternativa de espectrometria de massa por ioniza???o e dessor???o a laser assistida por matriz (MALDI-TOF) para a identifica???o de 36 isolados brasileiros de Aspergillus terreus. A esp??cie ?? importante mundialmente, dentre outros motivos, devido ?? habilidade de produzir lovastatina, uma droga bem sucedida na redu???o do colesterol plasm??tico com consequente redu???o do risco de doen??as cardiovasculares, principal causa mundial de ??bitos. Antes das an??lises por MALDI-TOF, o sequenciamento do espa??ador interno transcrito (ITS) permitiu a confirma???o da identidade de 94% (34/36) dos isolados quando comparado ?? base de dados GenBank. Inicialmente de forma a comparar a t??cnica de biologia molecular com a identifica???o por MALDI-TOF foi avaliado a influ??ncia de importantes fatores na qualidade dos dados e selecionado aqueles que mais se adequavam a identifica???o da esp??cie. Isso permitiu caracterizar quanto ao perfil proteico 30 dos 36 isolados. Contudo, somente 20% dos isolados brasileiros foram identificados como A. terreus quando comparado aos quase 4000 organismos da base de dados Biotyper o que pode ser atribu??do ?? baixa representatividade da esp??cie. Segundo os resultados existem fortes evid??ncias de que a t??cnica ?? adequada o que corrobora com isso ?? o padr??o semelhante observado nos dendrogramas gerados por cada uma das metodologias, a capacidade de distin???o dos isolados comparativamente maior por MALDI e a comprovada efic??cia da t??cnica avaliada pelo teste ??s escuras a partir da constru???o de uma biblioteca pr??pria. Por essas raz??es o emprego de MALDI-TOF na identifica???o de isolados de A. terreus em substitui???o as t??cnicas tradicionais ?? promissora, por??m requer otimiza???es no que diz respeito ?? padroniza???o quanto ao processamento das amostras e o incremento de mais espectros de refer??ncia da esp??cie no banco de dados.
