In VitroDevelopment and Analysis ofEscherichia coliandShigella boydiiAzithromycin–Resistant Mutants

Abstract

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-β-Naphthylamide (PAβN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAβN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAβN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…In a study on Salmonella enterica serovar Typhimurium with an inactivated acrB gene, Nikaido et al (1998) observed a 64-fold decrease in the MIC of erythromycin (from 512 mg/L to 8 mg/L). These observations are consistent with the aforementioned effect of the PAbN (Gomes et al, 2013b) and with the 128-fold decreases in the MIC levels to erythromycin, clarithromycin and azithromycin (from 512 mg/L to 4 mg/L for the first two and from 64 to 0.5 for azithromycin) described by Wehmeier et al (2009) when disrupting the acrB gene. As with other antimicrobial families, this kind of generic efflux pumps are not able to extrude all macrolide members from the bacterial cytoplasm, thereby demonstrating the inability of AcrAB-TolC to pump out telithromycin (Chollet et al, 2004).…”

“…The downexpression of OmpW has been observed in E. coli strains exhibiting resistance to ceftriaxone (Hu et al, 2005), while, as in the case of azithromycin, its overexpression has been observed in in vitro obtained tetracycline resistant E. coli strains (Zhang et al, 2008). Additionally, the overexpression of OmpW was previously observed in another spontaneous Shigella boydii azithromycinresistant mutant obtained in the absence of PAbN (Gomes et al, 2013b). These data suggest the role of OmpW as an extrusion way of azithromycin as has been proposed for other substances (Gil et al, 2007).…”

“…However, not all alterations in either 23S rRNA or the L4 and L22 proteins result in cross-resistance to all members of the macrolide family, with some being affected by specific alterations (Ettayebi et al, 1985). Chromosomal efflux pumps have been reported to have a relevant role in macrolides resistance (Gomes et al, 2013b;Wehmeier et al, 2009). In addition, a series of less studied chromosomal encoded mechanisms, such as the presence of short peptides encoded in "mini-genes" able to affect the susceptibility levels to macrolides have also been described Tripathi et al, 1998) despite not taking part in the development of macrolide resistance in clinical Enterobacteriaceae.…”

“…Thus, the real relevance of these efflux pumps is only visualized if they are inactivated or inhibited (Gomes et al, 2013c;S aenz et al, 2004), while their role in antimicrobial resistance at a clinical level is observed when they are overexpressed (Li et al, 2015). Efflux pump overexpression has been related to the development of azithromycin resistance in 19 E. coli azithromycin-resistant mutants obtained in vitro in which the use of PAbN, an inhibitor of RND-type efflux pumps, resulted in a fourto 16-fold decrease in the MIC (Gomes et al, 2013b). In E. coli and Shigella spp.…”

Characterization of two Achromobacter xylosoxidans isolates from patients with pertussis-like symptoms

“…The 23S rRNA gene was amplified from bacterial DNA extractions using the primers and conditions described previously [20]. Additionally, the rplV and rplD genes from both nasopharyngeal swabs and bacterial origins were amplified using Bordetella-specific primers: rplV-F:…”