Antimicrobial Susceptibility Studies
Diversity of polymyxin resistance mechanisms among Acinetobacter baumannii clinical isolates

https://doi.org/10.1016/j.diagmicrobio.2016.10.011Get rights and content

Highlights

  • Our results showed that the mechanisms of resistance to polymyxin B varied significantly between A. baumannii clinical isolates. In contrast to previously reported data, the A027ind strain had acquired simultaneously mutations in both pmrB and lpxA genes after polymyxin B exposure.

  • By transmission electronic microscopy, the exposure to polymyxin B induced a strong condensation and darkening of intracellular material of A. baumannii isolates.

  • The C. elegans assays showed that the A027 strain, which was initially susceptible to polymyxin B, was more virulent than polymixin-resistant A009 strain. However, after polymyxin B exposure, A027ind strain displayed a significant decrease of virulence, which was not observed in the A009ind strain.

Abstract

Polymyxins have become drugs of last resort for treatment of multi-drug resistant (MDR) Gram-negative infections. However, the mechanisms of resistance to this compound have not been completely elucidated. In this study, we evaluated the mechanisms of resistance to this antimicrobial in two A. baumannii clinical isolates, respectively, susceptible (A027) and resistant (A009) to polymyxin B before and after polymyxin B exposure (A027ind and A009ind). The pmrAB and lpxACD were sequenced and their transcriptional levels were analyzed by qRT-PCR. The bacterial cell morphology was evaluated by transmission electronic microscopy (TEM) and the membrane potential was measured using Zeta-potential analyzer. The virulence of strains was studied using a Caenorhabditis elegans model. Both clinical isolates exhibited an elevation of the polymyxin B MIC after exposure to this compound. On the other hand, A027ind showed decreased values of MIC for β-lactams, aminoglycosides, vancomycin, teicoplanin, oxacillin and erythromycin. A027ind harbored two mutations in pmrB and the ISAba125 disrupting the lpxA. In contrast, A009ind strain exhibited increase of pmrB transcriptional level, after polymyxin B exposure, despite the absence of mutations in the pmrAB genes. The TEM images revealed a thicker and more electron-dense peptidoglycan layer for A009 than that of A027. The exposure to polymyxin B induced a strong condensation and darkening of intracellular material, mainly in A009ind. In addition, the surface charge of A009 was significantly less negative than the one of A027. Using the C. elegans model, only A027ind strain showed a reduction on virulence. The diversity of polymyxin B resistance mechanisms among A. baumannii strains evaluated in this study confirms the complexity of these mechanisms, which may vary depending of the background of each strain.

Introduction

Acinetobacter baumannii ranked as the fourth most common pathogen causing catheter related bloodstream infections in adult patients hospitalized at Brazilian intensive care units (UTIs) (Brazilian Health Surveillance Agency (ANVISA), 2013). Its ability to adapt in adverse conditions, as occurred in the hospital environment, contribute to resistance development in this pathogen (Vila et al., 2007). According to the latest report of the Brazilian Health Agency, 80.7% of Acinetobacter spp. from ICUs was resistant to carbapenems. Similar rates have been observed in Europe and in the USA, where 90.6% and 63.0% of Acinetobacter spp. were reported as resistant to these compounds, respectively (Brazilian Health Surveillance Agency (ANVISA), 2013; Centers for Disease Control and Prevention (CDC), 2013; European Centre for Disease Prevention and Control). This phenotype has been mainly attributed to the spread of OXA-23-producing clones (Cardoso et al., 2016, Chagas et al., 2014, Gheorghe et al., 2015, Grosso et al., 2011, Merino et al., 2014, Vasconcelos et al., 2015, Werneck et al., 2011). For this reason, polymyxins have been frequently administered as empirical therapy for ICU patients diagnosed with ventilator-associated pneumonia. However, resistance to these drugs has already been reported, and the mechanisms involved in this phenotype are not fully understood (Marchaim et al., 2011, Snitkin et al., 2013). To date, two distinct mechanisms of polymyxins resistance have been characterized in A. baumannii. The first one involves the modification of the membrane lipopolysacharide (LPS) through addition of phosphoethanolamine in the lipid A moiety, in a process mediated by PmrAB two-component system. Single nucleotide mutations and/or increased expression of pmrA or pmrB leads to up-regulation of pmrC, which, in turn, produces phosphoethanolamine transferase, responsible for modification on the lipid A. This modification reduces the negative charge of the outer bacterial membrane and, therefore, decreases the polymyxin affinity for the bacterial cell surface (Adams et al., 2009, Beceiro et al., 2011, Cardoso et al., 2016, Marchaim et al., 2011, Park et al., 2011, Snitkin et al., 2013). Other mechanism of polymyxin resistance described in A. baumannii is the complete loss of LPS due to single nucleotide mutations on lpxA, a component of LPS biosynthesis operon (Moffatt et al., 2010). Mutations on the other genes of LPS biosynthesis pathway, such as lpxC and lpxD, have also been reported in polymyxin-resistant A. baumannii laboratory derivative strains (Moffatt et al., 2010, Wand et al., 2015). In addition, other mechanisms might be present among clinical strains since polymyxin-resistant strains that did not have any of the mechanisms described above have also been reported (Lee et al., 2015). Recently, a plasmid-mediated phosphoethanolamine transferase, denominated MCR-1, was reported among Escherichia coli and Klebsiella pneumoniae isolated from humans and pigs from China (Liu et al., 2016).

Several studies described decrease in the fitness of polymyxin-resistant Acinetobacter spp. strains due to energetic cost for in vitro adaptive resistance development (Beceiro et al., 2014, Fernández-Reyes et al., 2009, Hraiech et al., 2013, López-Rojas et al., 2011a, López-Rojas et al., 2013). Acinetobacter spp. strains showing this resistance phenotype usually display a slower growth rate compared with polymyxins susceptible ones. In contrast, Durante-Mangoni et al. recently reported the emergence of colistin resistance without loss of fitness and virulence in clinical extensively-multi drug resistant A. baumannii after prolonged colistin administration (Durante-Mangoni et al., 2015).

In this manner, this study was undertaken to evaluate the mechanism of polymyxin B resistance of two A. baumannii clinical isolates, initially identified as susceptible and resistant to polymyxins, before and after polymyxin B exposure. In addition, we also studied the fitness and virulence of each isolate using the Caenorhabditis elegans animal model.

Section snippets

Strains

Two A. baumannii clinical isolates, initially characterized as susceptible and resistant to polymyxins by the routine clinical laboratory, based on CLSI breakpoints (Clinical Laboratory Standard Institute (CLSI), 2015), were selected for this study, as described in the Table 1. The identification at the species level was confirmed by rpoB DNA-sequencing (La Scola et al., 2006). These clinical isolates were subcultured in Luria Bertani (LB) agar supplemented with increasing concentrations of

Results

Each pair of Acinetobacter strains was confirmed to be genetically identical by PFGE. The mutant strains recovered after polymyxin B exposure were genetically identical to their respective parental isolates. The Table 3 describes the antimicrobial susceptibility profile of A. baumannii clinical isolates and their mutants. A027 strain was initially susceptible only to polymyxin B (MIC, 0.25 μg/mL) and tigecycline (MIC, 1 μg/mL). After polymyxin B exposure, the A027ind had an elevation of the

Discussion

Resistance to carbapenems has drastically increased over decades worldwide. For instance, the imipenem resistance rates among A. baumannii clinical isolates increased from 12.6% to 71.4% between 1997 and 1998 and 2008–2010 periods in some Brazilian medical centers (Gales et al., 2012). Although novel antimicrobials have been recently approved by the Food and Drug Administration (FDA) for the treatment of Gram-negative infections, none of them are active against carbapenem-resistant A. baumannii

Transparency Declarations

A.C.G. has received research funding and/or consultation fees from Astra-Zeneca, MSD, and Novartis. Other authors have nothing to declare. This study has not been financially added by any Diagnostic/Pharmaceutical company. This work was presented in part as a poster (C1–1080) at the 53th Interscience Conference of Antimicrobial Agents and Chemotherapy – ICAAC in Denver, 2013.

Acknowledgments

We would like to thank the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology (Brazil), for providing a research grant to A.C.G. (305535/2014-5) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financially supporting this study (2010/12891-9) and granting the Post-Doctoral scholar fellowship to R.G. (2012/15458-0) and to R.C. (2012/15459-6).

We also thank the Electron Microscopy Center – UNIFESP, especially to Prof. Edna

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