Antimicrobial Susceptibility StudiesDiversity of polymyxin resistance mechanisms among Acinetobacter baumannii clinical isolates
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
References (45)
- et al.
Characterization of carbapenem-resistant Acinetobacter baumannii in Brazil (2008-2011): countrywide spread of OXA-23-producing clones (CC15 and CC79)
Diagn Microbiol Infect Dis
(2014) - et al.
Emergence of colistin resistance without loss of fitness and virulence after prolonged colistin administration in a patient with extensively drug-resistant Acinetobacter baumannii
Diagn Microbiol Infect Dis
(2015) - et al.
Antimicrobial resistance among gram-negative bacilli isolated from Latin America: results from SENTRY antimicrobial surveillance program (Latin America, 2008-2010)
Diagn Microbiol Infect Dis
(2012) - et al.
Lipopolysaccharide loss produces partial colistin dependence and collateral sensitivity to azithromycin, rifampicin and vancomycin in Acinetobacter baumannii
Int J Antimicrob Agents
(2015) - et al.
Ceftolozane/tazobactam and ceftazidime/avibactam: two novel β-lactam/β-lactamase inhibitor combination agents for the treatment of resistant gram-negative bacterial infections
Int J Antimicrob Agents
(2015) - et al.
Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study
Lancet Infect Dis
(2016) - et al.
Membrane permeabilization of colistin toward pan-drug resistant gram-negative isolates
Braz J Microbiol
(2016) - et al.
Correlation between overexpression and amino acid substitution of the PmrAB locus and colistin resistance in Acinetobacter baumannii
Int J Antimicrob Agents
(2011) - et al.
The changing epidemiology of Acinetobacter spp. producing OXA carbapenemases causing bloodstream infections in Brazil: a BrasNet report
Diagn Microbiol Infect Dis
(2015) - et al.
Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of staphylococcus pseudintermedius, Escherichia coli, Pseudomonas Aeruginosa and Malassezia pachydermatis?
Vet Microbiol
(2015)
Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system
Antimicrob Agents Chemother
New insights into the antibacterial mechanism of action of squalamine
J Antimicrob Chemother
Antibiotic resistance and its cost: is it possible to reverse resistance?
Nat Rev Microbiol
Phosphoethanolamine modification of lipid a in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system
Antimicrob Agents Chemother
Biological cost of different mechanisms of colistin resistance and their impact on virulence in Acinetobacter baumannii
Antimicrob Agents Chemother
Boletim Informativo: Segurança e qualidade em serviço de saúde n° 9: Relatório da resistência microbiana em infecções primárias de corrente sanguínea confirmadas laboratorialmente relacionadas ao uso de cateter venoso central em unidades de terapia intensiva
The genetics of Caenorhabditis elegans
Genetics
Diversity of mechanisms conferring resistance to β-lactams among OXA-23-producing Acinetobacter baumannii clones
Diagn Microbiol Infect Dis
Antibiotic resistance threats in the United States. Cdar-threats-2013-508
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard - ninth edition
Document M07-A9
Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement
Document M100-S25
Phytochemical and antibacterial investigations of the extracts and fractions from the stem bark of Hymenaea stigonocarpa Mart. Ex Hayne and effect on ultrastructure of Staphylococcus aureus induced by hydro alcoholic extract
Sci World J
Cited by (25)
Antimicrobial activity and biofilm inhibition of riparins I, II and III and ultrastructural changes in multidrug-resistant bacteria of medical importance
2020, Microbial PathogenesisCitation Excerpt :Thus, our findings with the activity of riparin III corroborate previous studies that observed the possible morphological changes induced in vitro by the action of antimicrobials. Other studies also describe numerous morphological changes in bacterial cells of S. aureus and A. baumannii when exposed to antimicrobials [33,34]. Our data corroborate these studies, since in the presence of riparin III it was possible to observe morphological changes in S. aureus and A. baumannii.
Molecular mechanisms of antimicrobial resistance in Acinetobacter baumannii, with a special focus on its epidemiology in Lebanon
2018, Journal of Global Antimicrobial ResistanceCitation Excerpt :Acquisition of colistin resistance is mainly due to modification in the lipopolysaccharide (LPS) biosynthesis pathways [17,23]. This modification decreases the negative charge of the cytoplasmic membrane and, consequently, reduces the affinity of colistin for the bacterial surface [89]. Two mechanisms of resistance to colistin have been described in A. baumannii: alteration of lipid A, a component of LPS, by several mutations affecting the two-component regulatory system PmrAB; and lack of production of LPS resulting from mutations in the genes lpxA, lpxC and lpxD that encode enzymes catalysing the first stages of LPS synthesis [90–92].