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Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

Nicolas M. Vazquez,Florencia Mariani,Pablo S. Torres,Silvia Moreno ,Estela M. Galván

Abstract

Escherichia coli is the most frequent agent of urinary tract infections in humans. The emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) has created additional problems in providing adequate treatment of urinary tract infections. We have previously reported the antimicrobial activity of 1,8-cineole, one of the main components of Rosmarinus officinalis volatile oil, against Gram negative bacteria during planktonic growth. Here, we evaluated the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of MDR ESBL-producing uropathogenic E. coli clinical strains by carrying out different technical approaches such as counting of viable cells, determination of biofilm biomass by crystal violet staining, and live/dead stain for confocal microscopy and flow cytometric analyses. The plant compound showed a concentration- and time-dependent antibiofilm activity over pre-formed biofilms. After a 1 h treatment with 1% (v/v) 1,8-cineole, a significant decrease in viable biofilm cell numbers (3-log reduction) was observed. Biofilms of antibiotic-sensitive and MDR ESBL-producing E. coli isolates were sensitive to 1,8-cineole exposure. The phytochemical treatment diminished the biofilm biomass by 48–65% for all four E. coli strain tested. Noteworthy, a significant cell death in the remaining biofilm was confirmed by confocal laser scanning microscopy after live/dead staining. In addition, the majority of the biofilm-detached cells after 1,8-cineole treatment were dead, as shown by flow cytometric assessment of live/dead-stained bacteria. Moreover, phytochemical-treated biofilms did not fully recover growth after 24 h in fresh medium. Altogether, our results support the efficacy of 1,8-cineole as a potential antimicrobial agent for the treatment of E. coli biofilm-associated infections.

Introduction

Uropathogenic Escherichia coli is the most common cause of urinary tract infections, accounting for approximately 80% of infections. The routine therapy of urinary tract infections is based on the use of antibiotics such as β-lactams, trimethoprim, nitrofurantoin and quinolones in many countries. Over-use and misuse of these antibiotics increase the development of resistant bacteria. Particularly, the emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) is a serious global health problem, since it can cause prolonged hospital stay, increasing morbidity, mortality, and health care costs. ESBLs are a group of β-lactamase enzymes that confer resistance to third generation cephalosporins, such as ceftazidime and ceftriaxone. Resistance genes coding for β-lactamases are often located on plasmids which also harbor resistance genes for non- β-lactam antibiotics such as aminoglycosides and trimethoprim-sulfamethoxazole. Therefore, ESBL producing bacteria are commonly MDR, leaving limited antibacterial options.

Materials & Methods

Bacterial strains and inoculum preparation

Escherichia coli strains used in this study were isolated from adult patients and are described in Table 1. E. coli strains named Ec AM were isolated from urinary samples collected from patients admitted to a medical center at Buenos Aires (Argentina) between 2017 and 2018 [16]. Strain Ec07 was isolated from a patient with polymicrobial CAUTI at Hospital Pirovano (Buenos Aires City, Argentina) [17]. Microbiological identification and antimicrobial susceptibility testing were carried out by standard methods. In vitro susceptibility tests were interpreted based on CLSI breakpoints [18]. E. coli strains were examined for ESBL production by a double-disk synergy test using ceftazidime, cefotaxime and cefepime with and without clavulanic acid according to CLSI guidelines [18]. E. coli clinical strains used in this study were isolated as part of routine clinical hospital procedures to diagnose infection and hence ethical approval was not required, according to the corresponding institutional guidelines.

Discussion

The extended biofilm recalcitrance toward antibiotic treatment has generated an urgent need for novel strategies against biofilm-associated infections [5]. We have previously reported that 1,8-cineole exhibits bactericidal activity against planktonic E. coli cells [12]. Therefore, we analyzed here the antibiofilm activity of this phytochemical against MDR ESBL-producing uropathogenic E. coli strains that are biofilm producers.

Acknowledgments

We are grateful to Claudia Garbasz, Head of the Microbiology Service at the Hospital General de Agudos “Dr. I. Pirovano” (Buenos Aires city, Argentina) for providing the clinical bacterial isolates. We are also thankful to Dr. Marisa Gomez and Dr. Camila Ledo for help with flow cytometry experiments. NMV and FM are doctoral fellows and PST, SM and EMG are researcher members of CONICET.

Citation: Vazquez NM, Mariani F, Torres PS, Moreno S, Galván EM (2020) Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole. PLoS ONE 15(11): e0241978. https://doi.org/10.1371/journal.pone.0241978

Editor: Monica Cartelle Gestal, University of Georgia, UNITED STATES

Received: February 13, 2020; Accepted: October 25, 2020; Published: November 5, 2020

Copyright: © 2020 Vazquez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability:

All files are available from the Figshare database (https://doi.org/10.6084/m9.figshare.c.5064746.v3).

Funding:

This work was supported by grant number PIP 11220130100426CO from the National Research Council of Argentina (CONICET) to EMG; grant number PICT 2017-0183 from Agencia Nacional de Promocion Cientifica y Tecnologica, Argentina, to EMG; and intramural funding from Fundacion Cientifica Felipe Fiorellino, Universidad Maimonides, Argentina, to SM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:

The authors have declared that no competing interest exist.

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