A virulence associated siderophore importer causes antimicrobial efflux in Klebsiella pneumoniae

The accessory genome of many pathogenic bacteria includes ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognised structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the sensitivity of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and involved antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognised as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its sensitivity to a broad range of antimicrobials. Thus, a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

The accessory genome of many pathogenic bacteria includes ABC transporters that scavenge metal by 17 siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug 18 efflux. There are mechanistic and recently recognised structural similarities between siderophore 19 importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ 20 on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster 21 in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of 22 Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp 23 increased the sensitivity of K. pneumoniae to all antimicrobials tested. The mechanism was dependent 24 on the yersiniabactin importer YbtPQ and involved antimicrobial efflux, since it was affected by the 25 inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. 26 pneumoniae is recognised as a reservoir of genes for the emergence of hospital outbreak strains and 27 for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, 28 to E. coli decreased its sensitivity to a broad range of antimicrobials. Thus, a confirmed siderophore 29 importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have 30 a secondary function exporting antimicrobials. 31

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Introduction 32 ABC transporters in the accessory genomes of bacterial pathogens significantly influence both 33 virulence and antimicrobial resistance. Siderophore importers scavenge metals from the host and efflux 34 pumps export antimicrobials, and the presence of such transporters on mobile genetic elements is 35 associated with both disease severity and treatment failure. 36 The nature of this transport is specific and unidirectional, due to the specific interactions between 37 substrate and binding cavity, and the asymmetry of ATP-powered conformational changes through 38 inward facing, closed and outward-facing forms. Broad-specificity multidrug efflux pumps are an 39 apparent exception, and these have binding cavities with multiple sites that can interact with diverse 40 antimicrobials [1]. Other reported exceptions include siderophore export by multidrug efflux pumps 41 and antibiotic entry through an asparagine importer [2,3]. By contrast, bifunctional ABC transporters 42 that import one substrate and export another, are compatible with the mechanistic models but are 43 unknown. 44 Siderophore importers are plausible candidates for such bidirectional transport since they have a 45 spacious substrate binding cavity that might accommodate other molecules, and the structural 46 organisation is exporter-like [4,5]. Furthermore, in the context of hospital outbreak strains, a secondary 47 function in antimicrobial export could provide a selective advantage. 48 The yersiniabactin siderophore cluster is prevalent and spreading in Klebsiella pneumoniae [6,7] and 49 was found in integrative and conjugative elements in approximately half the K. pneumoniae clinical 50 isolates tested in a recent UK and global study [8]. This cluster is also common in pathogenicity islands 51 in We used genetic manipulation to investigate whether YbtPQ influences antimicrobial susceptibility of 60 K. pneumoniae and E. coli. We found that the transporter was necessary and sufficient to confer a 61 modest but significant reduction in sensitivity to a broad range of antimicrobials (all antimicrobials 62 tested). The effect was due to antimicrobial efflux, since it was blocked by the efflux pump inhibitor 63 reserpine. 64 2 Results 65 2.1 ICEKp and its transporter gene cluster influence the antimicrobial sensitivity of K. 66 pneumoniae 67 The influence of ICEKp and cargo genes on antimicrobial susceptibility was investigated by measuring 68 growth inhibition of K. pneumoniae by antimicrobials in liquid medium (minimum inhibitory 69 concentration assay) and agar (zone of inhibition assay pSXPQA was constructed to reintroduce the yersiniabactin transporter gene cluster (five genes), and 74 this plasmid restored the MIC to parental levels for all antimicrobials (table 1). The second method, 75 disc diffusion assay, was used to confirm the change in antimicrobial sensitivity and test new classes 76 of antimicrobial ( figure 1). For all antimicrobials tested, ICE was more sensitive than KpRR2 77 (significantly larger zone of inhibition, p<0.05, figure 1) whereas the plasmid-complemented mutant 78 was not significantly different from the parent strain. 79

ICEKp and the transporter gene cluster reduce antimicrobial sensitivity by enhancing 80
antimicrobial efflux 81 ABC transporters that efflux antimicrobials can be blocked by the inhibitor reserpine [20,21]. To 82 determine whether ICEKp affects antimicrobial sensitivity by causing efflux of antimicrobials, we 83 repeated the disc diffusion assay using reserpine (figure 2). Reserpine significantly enhanced the 84 sensitivity of the parental strain and the complemented strain (p<0.5) but not the mutant strain, thus 85 effects of ICEKp and the transporter plasmid are due to an efflux mechanism. 86 conformation of the YbtPQ transporter is consistent with antimicrobial access to the cavity, which 116 could lead to transport by the measured basal ATPase activity, or by antimicrobial-enhancement of 117 ATP binding and hydrolysis [17]. Apart from the putative efflux activity of YbtPQ, alternative 118 explanations for its effect on antimicrobial sensitivity could include YbtPQ-induced changes in gene 119 expression or YbtPQ-catalysed transport of other molecules. 120

The ABC transporter
The yersiniabactin cluster is one of the key virulence associated factors reported in surveillance studies 121 of outbreaks and spread of K. pneumoniae and here we demonstrate a potential selective advantage for 122 this cluster in the presence of antimicrobials. Given that expression of the cluster is upregulated in 123 infection models, this advantage could be significant in patients infected with K. pneumoniae who are 124 receiving antibiotic treatment. 125 Other siderophore transporters from related or unrelated clusters might similarly influence 126 antimicrobial resistance. The virulence enhancing piscibactin plasmids, carrying a YbtPQ homologue, 127 are transmissible between species and enhance virulence of the economically fish aquaculture 128 pathogen, Photobacterium damselae subsp piscidia [23,24]. 129 The facile movement of a virulence determinant from a multidrug resistant clinical isolate to another 130 Gram-negative bacterium, with associated reduction in antimicrobial susceptibility, highlights the 131 complex threat posed by the evolution and spread of drug resistance loci and drug resistant pathogens. 132 4 Methods 133 Plasmid pSXPQA contained the native promoter, whereas the genes in the other three plasmids were 150 cloned under the tetracycline promoter. Plasmid construction was verified by sequencing. 151

Antibiotic susceptibilities by disc diffusion and E-test method 152
Colonies from overnight LA plate cultures were picked and suspended in sterile 0.9 % NaCl. The 153 turbidity of the suspension was adjusted to match McFarland 0.5 standard (~0.8 at OD600). The 154 suspension was spread evenly on MHB agar within 15 min of preparation using a sterile cotton swab 155 to create a semi-confluent growth. The inoculum was allowed to dry for 10 min before applying the 156 antibiotic discs or Etest strips then the plates were incubated at 37 o C for 24 hours. Any plates with 157 uneven growth were discarded. The zone of growth inhibition around the discs (Oxoid) was measured 158 in mm and results are reported as the mean and standard deviation of three or more independent 159 replicates. For Etest (BioMerieux), the MIC (μg/ml) was read from the scale on the Etest strip where 160 the symmetrical inhibition ellipse edge intercepts the strip and results are reported as the mean of five 161 independent replicates. 162  Transconjugant E. coli+ICE carrying the ICEKp was significantly less sensitive than E. coli to 194 trimethoprim and tetracycline in a disc diffusion assay (25 g and 10 g per disc The raw data supporting the conclusions of this article will be made available by the authors, without 219 undue reservation. 220 11 References 221