A multidrug resistant clinical P. aeruginosa isolate in the MLST550 clonal complex: uncoupled quorum sensing modulates the interplay of virulence and resistance

Abstract

22Pseudomonas aeruginosa is a prevalent and pernicious pathogen equipped with both extraordinary 23 capabilities to infect the host and to develop antimicrobials resistance (AMR). Monitoring the 24 emergence of AMR high risk clones and understanding the interplay of their pathogenicity and 25 antibiotic resistance is of paramount importance to avoid resistance dissemination and to control 26 P. aeruginosa infections. In this study, we report the identification of a multidrug resistant (MDR) 27 P. aeruginosa strain PA154197 isolated from a blood stream infection in Hong Kong. PA154197 28 belongs to a distinctive MLST550 clonal complex shared by two international P. aeruginosa 29 isolates VW0289 and AUS544. Comparative genome and transcriptome analysis with the 30 reference strain PAO1 led to the identification of a variety of genetic variations in antibiotic 31 resistance genes and the hyper-expression of three multidrug efflux pumps MexAB-OprM, 32 MexEF-OprN, and MexGHI-OpmD in PA154197. Unlike many resistant isolates displaying an 33 attenuated virulence, PA154197 produces a significantly high level of the P. aeruginosa major 34 virulence factor pyocyanin (PYO) and displays an uncompromised virulence compared to PAO1. 35 Further analysis revealed that the secondary quorum sensing system Pqs which primarily controls 36 the PYO production is hyper-active in PA154197 independent of the master QS systems Las and 37 Rhl. Together, these investigations disclose a unique, uncoupled QS mediated pathoadaptation 38 mechanism in clinical P. aeruginosa which may account for the high pathogenic potentials and 39 antibiotics resistance in the MDR isolate PA154197. 40 41 Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen that causes a variety of 42 notorious infections in humans such as ventilator-associated pneumonia, lung infections of cystic 43 fibrosis (CF) patients, burn wound infection, and various sepsis syndromes. It is the second leading 44 cause of hospital-acquired infections and is especially problematic in ICUs, where it is the leading 45 cause of pneumonia among pediatric patients and is responsible for a large number of urinary tract 46 (10% in US and 19% in Europe), blood stream (3% in US and 10% in Europe), eye, ear, nose, and47 throat infections (1-3). Compounding the burden of these infections is the extraordinary capability 48 of the pathogen to develop antibiotic and multidrug resistance (MDR) even during the course of 49 antibiotics therapy. P. aeruginosa is one of the "ESKAPE" (Enterococcus spp., Staphylococcus 50 aureus, Klebsiella spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter 51 spp.) organisms which are recognized by the WHO as an alarming threat to the global public heath 52 associated with antimicrobial resistance (AMR). As a consequence, the diseases outcome of the P. 53 aeruginosa infections is the complex interplay of the pathogen (its pathogenicity and virulence), 54 hospital environments (antibiotic therapies and the emergence of AMR), and the patie...

Native CRISPR-Cas mediated in situ genome editing reveals the exquisite interplay of resistance mutations in clinical multidrug resistant Pseudomonas aeruginosa

“…Three multidrug efflux pumps MexAB-OprM, MexEF-OprN, MexGHI-OpmD were found to be hyper-expressed in PA154197 compared to PAO1 [26]. To test their contribution to the MDR phenotype of the strain, we first delete mexB , mexF , and mexH , which encodes the inner membrane channel of the three efflux systems, respectively.…”

“…Analysis of the growth curves of these strains in the absence of antibiotics suggests that the observed MIC alterations are not due to intrinsic disadvantages or defects in growth (S5 Fig). Unexpectedly, deletion of another efflux gene mexH which is also hyper-expressed in PA154197 (~40-fold higher than in PAO1) [26] does not lead to any detectable difference in the MICs of all the antibiotics and antimicrobial agents tested (Fig 2 and S2 Table). Further deletion of mexH in the Δ mexB Δ mexF strain yields no MIC change either (data not shown), suggesting that it does not contribute to the antibiotic resistance in PA154197.…”

“…E. coli DH5α is used for plasmid construction and is usually cultured at 37°C in Luria-Bertani (LB) broth or on the LB agar plate supplemented with 20 μg/ml Kanamycin (KAN). P. aeruginosa PA154197 was isolated from the Queen Mary Hospital in Hong Kong, China [26]. PA154197 and its derivatives were selected in LB (broth or agar) with 500 μg/ml KAN at 37°C.…”

“…Previously, we have isolated a MDR P. aeruginosa strain PA154197 which displays high epidemic potentials with a resistance profile (resistant to five of the seven commonly used antipseudomonal drugs) comparable to the international high-risk clone ST175 [26]. A large number of resistant mutations were predicted by genomic analysis, including mutations in the DNA gyrase gyrA and Cephalosporinase ampC which are associated with resistance to fluoroquinolones (FQ) and β-lactams [27, 28], respectively, and gene mutations potentially causing over-production of three multidrug efflux pumps MexAB, MexEF, and MexGHI (S1 Table).…”

mlDEEPre: Multi-Functional Enzyme Function Prediction With Hierarchical Multi-Label Deep Learning

“…We believe that the idea of mlDEEPre, combining multi-label learning with deep learning, can be helpful for solving other similar bioinformatics problems. For example, it has the potential to be applied to predict the properties of antibiotic-resistant genes (ARG) in multidrug-resistant pathogens (Zhu et al, 2013; Cao et al, 2018), perform classification of multicomponent transporter system (Saier et al, 2015), and predict CRISPR-Cas9 gene editing off-target regions (Fu et al, 2013; Pattanayak et al, 2013; Lin and Wong, 2018; Zhang et al, 2018).…”