Tetracycline resistance in clinical and environmental isolates of Escherichia coli, Klebsiella pneumoniae and Aeromonas spp.: identification and mapping of tet genes genetic context

Author(s)

Livia Carminato Balsalobre

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Saúde Pública (FSP/CIR)
• Defense date: 2014-09-30

Examining board members

Maria Helena Matte; Ana Cristina Gales; Nilton Erbet Lincopan Huenuman; Maria Tereza Pepe Razzolini; Maria Inês Zanoli Sato

• Advisor: Maria Helena Matte

Abstract

Introduction. The antibiotic resistance is accepted as one of the major problems for public health. Tetracyclines are broad spectrum antibiotics, and its indiscriminate use promoted the emergence of resistant bacteria, leading physicians and veterinarians to decrease its use. Objectives. Verify the susceptibility of clinical and environmental isolates of Escherichia coli, Klebsiella pneumoniae and Aeromonas spp. to tetracyclines, and also search for the main tet genes associated with resistance to these antibiotics and determine the potential mechanism of tet genes dissemination by characterizing their genetic context. Material and Methods. Disk-Diffusion and Minimum Inhibitory Concentration tests were carried out in 572 isolates using tetracycline (TET), doxycycline (DOX), minocycline (MIN) and tigecycline (TGC). PCR was carried out in TET non-susceptible isolates for the detection of Inc groups, tet genes and its genetic context determination through the search of classes 1, 2, 3, and 4 integrases, and Tn1721, Tn10, IS26 and ISAS5 mobile genetic elements. Genetic similarities patterns were determined by ERIC-PCR and PFGE techniques. After analyzing the results 33 strains were selected for the S1-PFGE and transformation experiments. Results. From 572 isolates, 18.5 per cent were TET-resistant, 13.5 per cent DOX-resistant, 8 per cent MIN-resistant and none resistant to TGC. Twenty-two per cent and 16.3 per cent of clinical and environmental isolates were TET-resistant, in that order. Genes tet(A), tet(B), tet(C), tet(D) and tet(E), coding for efflux pump mechanism, were found in 25.5 per cent , 33 per cent , 6.5 per cent , 18.9 per cent and 23.5 per cent of the isolates, respectively. Ninety-five per cent, 100 per cent , 100 per cent and 4.5 per cent of the isolates carrying tet(A), tet(B), tet(D) and tet(E) were non-susceptible to DOX, respectively. Resistance to MIN was observed in 4.2 per cent , 78.8 per cent and 100 per cent of isolates carrying tet(A), tet(B) and tet(D), in that order. The gene tet(A) was associated with Tn1721, tet(B) with Tn10, and tet(C) and (D) with IS26. None of the searched integrases were associated with the tet genes detected. Groups IncF, IncFIB and IncA/C were respectively observed in 54.8 per cent , 41.1 per cent and 28.7 per cent of the isolates. One Aeromonas spp. was carrying an IncP plasmid. The genetic similarities patterns demonstrated that there were identical genetic patterns among the hospital K. pneumoniae isolates, however all the remaining isolates possessed distinct genetic patterns. Of the 33 strains selected for plasmid linearization and transformation experiments, 8 were successfully transformed, in which the presence of tet genes in plasmids were observed. Conclusions. A low level of tetracycline resistance was detected. TGC was the most active tested antibiotic, followed by MIN. Genes tet(A) and tet(B) were the most prevalent among the isolates. All strains carrying tet(B) and tet(D) were non-susceptible to DOX and MIN. Groups IncF, IncFIB and IncA/C were the most detected in this study. The results suggest that tet(A), (B), (C) and (D) are disseminated by plasmids and are associated with Tn1721, Tn10 and IS26. Additional studies assembling recent isolates and other genera are necessary in order to contribute with information about the bacteria resistance to tetracyclines. (AU)

• FAPESP's process: 10/08695-0 - Occurrence of tetracycline phenotypic and genotypic resistance in Aeromonas hydrophila, Escherichia coli e Klebsiella pneumoniae isolated from clinical and environmental sources
• Grantee: Livia Carminato Balsalobre
• Support Opportunities: Scholarships in Brazil - Doctorate