RESUMEN
BACKGROUND: Pseudomonas aeruginosa is an important causative agent of nosocomial infections. As pathogen, P. aeruginosa is of increasing clinical importance due to its ability to develop high-level multidrug resistance (MDR). METHODS: The aim of the present study was to better understand the intrinsic virulence of circulating strains of Pseudomonas aeruginosa, by surveying and characterizing the antibiotic resistance profiles and prevalence of virulence factors in 51 clinical isolates of P. aeruginosa obtained from children admitted to Hospital del Niño-Panamá during the period of October 2016 until March 2017. Antimicrobial susceptibilities were assessed by determining the minimum inhibitory concentration for 12 antibiotics against P. aeruginosa clinical isolates using the VITEK system (https://www.biomerieux.com). Additionally, all isolates were examined by Polymerase Chain Reaction (PCR) for the presence of components of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes and betalactamases resistance genes (ESBL) using gene-specific primers. RESULTS: A total of 51 pyoverdine producing clinical isolates were analyzed, all of which expressed resistance genes such as genes of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes (fpvA). Out of 51 MDR isolates, 22 were ESBL producers. The most common ESBL gene was blaTEM expressed by 43% of the isolates. The isolates tested in this study showed increased resistance to antibiotics in the following categories: (i) penicillins (ampicillin (69%), piperacillin (22%); (ii) pyrimethamines (trimethoprim, 65%); (iii) nitrofurans (nitrofurantoin, 63%), and (iv) third-generation cephalosporin cefotaxime (53%). These results underscore a high prevalence of MDR amongst clinical isolates from Panama. CONCLUSIONS: The present study indicates that prevalence of BlaTEM-carrying strains is increasing with subsequent multidrug resistance in Panamá and as well reported worldwide. The virulent factors identified in this study provide valuable information regarding the prevalence of resistance genes and their potential impact on treatments that exploit the unique physiology of the pathogen. To prevent further spread of MDR, the proportions of resistant strains of Pseudomonas aeruginosa should be constantly evaluated on healthcare institutions of Panamá. More importantly, this information can be used to better understand the evolution and dissemination of strains hoping to prevent the development of resistance in Pseudomonas aeruginosa. Future studies quantifying the expression of these virulent genes will emphasize on the acquisition of multidrug resistance.
Asunto(s)
Infección Hospitalaria , Infecciones por Pseudomonas , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/farmacología , Niño , Farmacorresistencia Bacteriana Múltiple/genética , Hospitales , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/farmacología , Pruebas de Sensibilidad Microbiana , Panamá , Prevalencia , Infecciones por Pseudomonas/epidemiología , Pseudomonas aeruginosa/genéticaRESUMEN
Pseudomonas aeruginosa produces, under conditions of iron limitation, a high-affinity siderophore, pyoverdine (PVD), which is recognized at the level of the outer membrane by a specific TonB-dependent receptor, FpvA. So far, for P. aeruginosa, three different PVDs, differing in their peptide chain, have been described (types I-III), but only the FpvA receptor for type I is known. Two PVD-producing P. aeruginosa strains, one type II and one type III, were mutagenized by a mini-TnphoA3 transposon. In each case, one mutant unable to grow in the presence of the strong iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA) and the cognate PVD was selected. The first mutant, which had an insertion in the pvdE gene, upstream of fpvA, was unable to take up type II PVD and showed resistance to pyocin S3, which is known to use type II FpvA as receptor. The second mutant was unable to take up type III PVD and had the transposon insertion in fpvA. Cosmid libraries of the respective type II and type III PVD wild-type strains were constructed and screened for clones restoring the capacity to grow in the presence of PVD. From the respective complementing genomic fragments, type II and type III fpvA sequences were determined. When in trans, type II and type III fpvA restored PVD production, uptake, growth in the presence of EDDHA and, in the case of type II fpvA, pyocin S3 sensitivity. Complementation of fpvA mutants obtained by allelic exchange was achieved by the presence of cognate fpvA in trans. All three receptors posses an N-terminal extension of about 70 amino acids, similar to FecA of Escherichia coli, but only FpvAI has a TAT export sequence at its N-terminal end.