Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of proteus vulgaris

Highlights

• Mechanism of colistin resistance in the frequent pathogen Proteus vulgaris is unknown.

• Role of aminoarabinose in colistin resistance in P. vulgaris is demonstrated.

• WGS and in vitro analysis of an atypical strain improve knowledge of this species.

• Highlights the potential role of some genes in colistin resistance.

Abstract

Colistin has become a last-line antibiotic for the treatment of multidrug-resistant bacterial infections; however, resistance to colistin has emerged in recent years. Some bacteria, such as Proteus and Serratia spp., are intrinsically resistant to colistin although the exact mechanism of resistance is unknown. Here we identified the molecular support for intrinsic colistin resistance in Proteus spp. by comparative genomic, transcriptomic and proteomic analyses of colistin-susceptible (CSUR P1868_S) and colistin-resistant (CSUR P1867_R) strains of an atypical Proteus vulgaris. A significant difference in outer membrane glycoside structures in both strains that was corroborated by MALDI-TOF/MS analysis was found, which showed an absence of 4-amino-4-deoxy-l-arabinose (L-Ara4N) in the outer membrane lipid A moiety of the susceptible strain. Comparative genomic analysis with other resistant strains of P. vulgaris available in a local database found a mutation in the arnBCADTEF operon of the susceptible strain. Transcriptomic analysis of genes belonging to the arnBCADTEF operon showed a significant decrease in mRNA expression level of these genes in the susceptible strain, supporting addition of L-Ara4N in the outer membrane lipid A moiety as an explanation for colistin resistance. Insertion of the arnD gene that was suggested to be altered in the susceptible strain by in silico analysis led to a 16-fold increase of colistin MIC in the susceptible strain, confirming its role in colistin resistance in this species. Here we show that constitutive activation of the arn operon and addition of L-Ara4N is the main molecular mechanism of colistin resistance in P. vulgaris.

Introduction

Antibiotic resistance has been a major public health concern worldwide for a number of years. Increased resistance, especially in Gram-negative bacteria (GNB), has forced clinicians to use more powerful antibiotics in the therapeutic arsenal, namely broad-spectrum antibiotics, especially carbapenems [1]. Unfortunately, resistance to carbapenems has appeared and has spread worldwide [1]. Facing this therapeutic impasse, clinicians have revived old antibiotics for the treatment of multidrug-resistant bacterial infections [2], whose use had been abandoned for decades owing to their toxicity, e.g. colistin [3]. Colistin is a polycationic antimicrobial peptide of the polymyxin family, belonging to the polymyxin E group, which is effective against numerous GNB. It acts as a detergent on cell membranes of GNB, displacing divalent cations that maintain the electrolytic balance of the cell membrane, causing disruption of the outer cell membrane and bacterial lysis. Charges in the cell membrane consist of phosphate groups that are present in the phospholipids and lipopolysaccharide (LPS), which mainly compose the cell membrane of GNB [3], [4]. Inevitably, the use of colistin over the past 10 years has led to the emergence of colistin resistance, detected in Pseudomonas spp. [5], Klebsiella pneumoniae [6], [7] and Acinetobacter spp. [7]. The mechanisms of colistin resistance are complex, as bacteria have developed numerous defence systems against antimicrobial peptides over a long time [8]. The mechanisms have mainly been studied in the case of acquired resistance, which explains part of the resistance to colistin in Enterobacteriaceae [9], [10]. The primary colistin resistance mechanism is synthesis of sugars such as phosphoethanolamine (pEtN) or 4-amino-4-deoxy-l-arabinose (L-Ara4N), which neutralise the negative charge of the cell membrane by binding the phosphate groups present on LPS, preventing the action of colistin [10]. This synthesis is regulated by many regulators, some of which are two-component systems such as PmrA/PmrB and PhoP/PhoQ, which are themselves regulated by other regulatory genes [9]. Recently, a pEtN transferase encoded by the mcr-1 gene has been found on conjugative plasmids in Escherichia coli [11], showing for the first time that colistin resistance is not only due to chromosomal mutations but may be transferable between bacteria [11].

Some GNB are intrinsically resistant to colistin, including Proteus, Providencia and Morganella spp. [9], [10]. In parallel with the emergence of acquired resistance in Enterobacteriaceae, an increase in infections due to Proteus, Providencia and Morganella spp. has been reported in several countries, including Argentina [12], the USA [13], Greece [14] and, more recently, France [15]. However, the molecular support for the intrinsic resistance to colistin in these bacteria remains largely unknown. Proteus spp. bacteria are widely present in the environment and have virulence factors such as flagella, fimbriae, enzymes (urease, proteases), haemolysins and endotoxins that allow them to colonise catheters [16]. They are mainly responsible for urinary tract infections and wound infections in humans [17]. Proteus mirabilis and Proteus vulgaris are the two original members of the genus Proteus and they are also the most commonly found in human infections [16], [17]. Studies on P. mirabilis have shown that its LPS contains L-Ara4N in the lipid A component [18], [19] as well as in its core region [20], which also may contain pEtN [20], [21], [22]. It has also been demonstrated that genes involved in L-Ara4N biosynthesis or regulation can modulate polymyxin resistance in this bacterium [10]. Here we studied an atypical clinical strain of P. vulgaris isolated from a urine sample that was abnormally susceptible to colistin. This study aimed to identify the molecular support for the intrinsic resistance to colistin in P. vulgaris by comparative genomic, transcriptomic and proteomic analysis of two strains of P. vulgaris (one susceptible and one resistant to colistin) and by in vitro tests allowing morphological and phenotypic differences of this atypical isolate to be highlighted.