A Potential High-Risk Clone of Pseudomonas aeruginosa ST463

Pseudomonas aeruginosa is one of the most common opportunistic pathogens, which causes severe nosocomial infections because of its well-known multidrug-resistance and hypervirulence. It is critical to curate routinely the epidemic P. aeruginosa clones encountered in the clinic. The aim of the present study was to investigate the connection between virulence factors and antimicrobial resistance profiles in epidemic clones. Herein, we found that ST463 (O4), ST1212 (O11), and ST244 (O5) were prevalent in 30 isolates derived from non-cystic fibrosis patients, based on multilocus sequence type (MLST) and serotype analysis. All isolates were multidrug-resistant (MDR) and each was resistance to at least three classes of antibiotics in antimicrobial susceptibility tests, which was consistent with the presence of the abundant resistance genes, such as blaOXA–50, blaPAO, aph(3′), catB7, fosA, crpP, and blaKPC–2. Notably, all blaKPC–2 genes were located between ISKpn6-like and ISKpn8-like mobile genetic elements. In addition, classical exotoxins encoded by exoU, exoS, and pldA were present in 43.44% (13/40), 83.33% (25/30), and 70% (21/30) of the isolates, respectively. The expression of phz operons encoding the typical toxin, pyocyanin, was observed in 60% of isolates (18/30) and was quantified using triple quadrupole liquid chromatograph mass (LC/MS) assays. Interestingly, compared with other MLST types, all ST463 isolates harbored exoU, exoS and pldA, and produced pyocyanin ranging from 0.2 to 3.2 μg/mL. Finally, we evaluated the potential toxicity of these isolates using hemolysis tests and Galleria mellonella larvae infection models. The results showed that ST463 isolates were more virulent than other isolates. In conclusion, pyocyanin-producing ST463 P. aeruginosa, carrying diverse virulence genes, is a potential high-risk clone.


INTRODUCTION
Pseudomonas aeruginosa is one of the most common gram-negative pathogens and is associated with ubiquitously acute and chronic infections, especially cystic fibrosis (Ji et al., 2013). The worldwide spread of P. aeruginosa poses a threat to global public health (Tacconelli et al., 2018). P. aeruginosa exhibits various mechanisms of antimicrobial resistance, including the use of efflux pumps, biofilm formation, an impermeable outer membrane, an adaptable genome, antibiotic-inactivating enzymes, mobile resistance genes, and target mutations (Curran et al., 2018;Horcajada et al., 2019;Zhu et al., 2019). Recently, the increasing incidence of multidrug resistance (MDR), particularly for carbapenems, has induced a new crisis involving nosocomial P. aeruginosa infections (Curran et al., 2018;Horna et al., 2019).
Various virulence factors have been demonstrated to contribute to P. aeruginosa infection. For example, type III effectors (exotoxins ExoS, ExoT, ExoY, and ExoU), type VI effectors (PldA), adherence factors (type IV pili, flagella), alginate, elastase, and biosurfactant rhamnolipid (Karatuna and Yagci, 2010;Boulant et al., 2018;Luo et al., 2019) play crucial roles in mortality (Juan et al., 2017). It should be noted that ExoU-positive P. aeruginosa is more likely to be resistant to multiple antibiotics, such as carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides (Hu et al., 2017), which further exacerbates infections and increases mortality (Horna et al., 2019). Interestingly, exoU has been reported to be mutually exclusive with exoS, a common gene in P. aeruginosa (Vareechon et al., 2017). Nevertheless, the coexistence of exoS and exoU enhances antibiotic resistance in P. aeruginosa (Horna et al., 2019). Moreover, pyocyanin, belonging to the family of phenazines, is the key virulence factor in P. aeruginosa. Pyocyanin is synthesized from chorismic acid through a series of biosynthetic enzymes encoded by the phz gene cluster (Supplementary Figure 1). Previous studies showed that pyocyanin can not only promote the pathogenicity to host cells by disrupting electron transport, cellular respiration, and energy metabolism (Rada and Leto, 2013), but also modulates bacterial physiology, such as survival, iron acquisition, biofilm formation, and antibiotic tolerance (Chincholkar and Thomashow, 2014;Zhu et al., 2019).
Recently, numerous epidemic P. aeruginosa strains have been described worldwide. For instance, ST175, ST235, and ST111 are high-risk clones with MDR profiles, among which ST235 is highly associated with exoU (Cholley et al., 2014). Infections caused by such strains often have a worse prognosis than infections with other strains. The combination of MDR and virulence factors always restricts the implementation of therapeutic options, thus there is an urgent need to investigate resistance and virulence characteristics to combat P. aeruginosa infections. The misuse and overuse of antibiotics, serving as a dominant driving force of resistance, might further shape the evolutionary trajectory of P. aeruginosa in the clinic and the environment. To date, the correlations between the presences of virulence factors, antibiotic resistance, and the genotype of P. aeruginosa in non-cystic fibrosis patients remain unclear. The present work investigated and characterized epidemic clones in a non-outbreak situation to shed light on the treatment options for P. aeruginosaassociated infections.

Bacterial Isolation
Thirty P. aeruginosa isolates were collected from 30 non-CF patients from the Second Affiliated Hospital of Zhejiang University School of Medicine from 2009 to 2018. The Second Affiliated Hospital of Zhejiang University School of Medicine is a general hospital with 3,200 beds, in which carbapenem-resistant P. aeruginosa (CRPA) had reached to 38.9% according to recent hospital surveillance. Thirty CRPA strains were randomly chosen from our previously sequenced genomes based on the sample source, isolation time, virulence factor, sequence type (ST), and carbapenemase genes. Specifically, the isolates were collected from sputum (n = 13), CVC (central vascular catheter, n = 3), blood (n = 3), urine (n = 3), feces (n = 3), pus (n = 4), and one sample with an unknown source. Detailed clinical information is shown in Supplementary Table 1. Before the experiments, all the isolates were re-identified using Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker Daltonics, Billerica, MA, United States).

Extraction of Pyocyanin
P. aeruginosa isolates were cultured on Luria-Bertani (LB) agar plates for 12 h, after which a single colony was selected for culture in LB broth at 37 • C with 200 rpm shaking for 16 h. After centrifugation 13,000 × g, the supernatant was collected, extracted twice with chloroform (5:3 v/v), and vortexed. The chloroform phase was kept after centrifugation (5,000 × g, 10 min) and mixed with 0.2 M HCl (3:1 v/v). The red phase was collected after centrifugation (5,000 × g, 10 min), extracted with onethird the volume of chloroform containing NaHCO 3 , and the chloroform phase (blue) was collected (El-Zawawy and Ali, 2016). The extract was dissolved with 90% acetonitrile for high performance liquid chromatography (HPLC)-mass spectrometry (MS) detection. The HPLC-MS apparatus (Shimadzu, HPLC/MS-8045, Kyoto, Japan) was equipped with a Shim-pack GIST-HP C18 column (2.1 mm × 50 mm, 3 µm, Shimadzu) at an oven temperature of 35 • C and a flow-rate of 0.3 mL/min. The gradient program was applied with the mobile phase consisting of solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1% formic acid in water) as follows: 95-70% of B for 0-5.00 min, 70-50% of B for 5.00-5.10 min, 50-30% of B for 5.10-7.10 min, 30-0% of B for 7.10-11.10 min, held at 0% B for 11.10-13.00 min, 0-95% of B for 13.00-14.00 min, and maintained at 95% B for 14.00-16.00 min. The positive electrospray ionization (ESI+) mode was chosen to analyze pyocyanin. The MS parameters for pyocyanin are shown in Supplementary  Table 2. The MS acquisition parameters used were as follows: gas temperature, 300 • C; drying gas, 10 L/min; heating Gas, 10 L/min; DL Temperature, 250 • C; heat block temperature, 400 • C; second pole collision gas, argon gas; and CID gas volt, 17 kPa.

Toxicity Evaluation
P. aeruginosa isolates were incubated in brain heart infusion (BHI) agar containing 5% sheep blood for hemolytic experiments. Virulence genes were analyzed by using BLAST software (SRST2 Toolkit version 0.2.0; Inouye et al., 2014), and the database of virulence genes at the NCBI. The virulence of P. aeruginosa isolates was evaluated in vivo using the Galleria mellonella larval infection model, and eight strains (1615( , 1802( , E211-2, 1608( , 1617( , 1104( , ZR16, and 1109 were selected to analyze their characteristics. Strains 1617 (ST1212), 1104 (ST244), ZR16 (ST463), and 1109 (ST463) are pyocyanin-producing isolates, and 1615 (ST1076), 1608 (ST1212), 1802 (ST1212), and E211-2 (ST274) are pyocyanin-negative isolates. PA14 was used as a reference strain for pyocyanin expression, and its mutant PA (phz genes cluster deleted) (Dietrich et al., 2013) was used as a negative control to evaluate the contribution of pyocyanin to the pathogenicity of P. aeruginosa. To prepare the inoculum, bacteria were grown for 12 h at 37 • C with 200 rpm shaking, washed in sterile phosphate-buffered saline (PBS) after centrifugation at 3,000 × g, and then adjusted to a final concentration of 10 5 colony forming units (CFU)/mL using a Nephelometer (Merieux, Nürtingen, Germany). A 10 µL aliquot of suspended strains (10 3 CFU of bacteria) was injected into each larva and incubated at 37 • C. Larvae were considered as dead if they did not respond to touch. The survival rates of G. mellonella larvae were recorded. The statistical analysis in this study is performed using GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, United States). Continuous variables were described using the mean ± SD and categorical variables as the number (percentage). T-tests were conducted to assess the normal distribution of continuous data, while the Chi-squared or Fisher's exact test were used to assess the categorical data. A P-value < 0.05 was considered statistically significant.

DNA Extraction and Genetic Analysis
Genomic DNA of all 30 isolates was extracted using a Wizard genomic DNA purification kit (Promega, Beijing, China) according to the manufacturer's instructions. The genomic DNA was then sequenced using the Illumina HiSeq X10 platform (Illumina, San Diego, CA, United States) with the 150-bp paired-end strategy. Raw reads were trimmed and assembled to contigs using SPAdes version 3.11.1 (Bankevich et al., 2012). Assembled contigs were analyzed via the Center for Genomic Epidemiology website to screen for the presence of acquired antimicrobial resistance genes (ARGs) 1 (Boolchandani et al., 2019). The multilocus sequence type (MLST) 2 (Larsen et al., 2012) and serotype 3 (Thrane et al., 2016) were also determined. Virulence-associated genes and mobile genetic elements (MGEs) were collected from the NCBI database and were identified using SRST2 Toolkit version 0.2.0. The genomes of the 27 pyocyanin-producing isolates were obtained from the Pseudomonas Genome Database 4 . The phylogenetic tree was analyzed using Parsnp in the Harvest package based on the core genome sequences of the eight P. aeruginosa ST463 strains (Schürch et al., 2018). The tree was then visualized using the online tool iTOL (Cui et al., 2020).
To explore whether there are any relationships between ST types and pyocyanin production in P. aeruginosa, we collected 27 genomes of pyocyanin-positive isolates from the Pseudomonas Genome Database (Supplementary Table 3.). We found that there was no common ST type that produced pyocyanin ( Table 2). Taken together, these results suggested a positive connection between pyocyanin production and ST463type P. aeruginosa strains.
To further evaluate the virulence potential of ST463, particularly the contribution of pyocyanin to survival rates, eight clinical isolates, 1615, 1802, E211-2, 1608, 1617, 1104, ZR16, and 1109 with different pyocyanin production levels, were used to challenge G. mellonella larvae. P. aeruginosa PA14, with high pyocyanin production and its mutant, P. aeruginosa PA, with the deletion of pyocyanin producing phz genes (Zhu et al., 2019), were used as reference strains. We observed that the isolates that produced high levels of pyocyanin (P. aeruginosa 1617, 1104, ZR16, and 1109) induced higher mortality than those without pyocyanin production (P. aeruginosa 1615, 1802, E211-2, and 1608) ( Figure 2D). This was in agreement with the finding that P. aeruginosa PA14 was more toxic to the larvae than P. aeruginosa PA, indicating that pyocyanin plays an important role in the pathogenicity of P. aeruginosaassociated infections. Notably, the ST463 type isolates (P. aeruginosa ZR16 and 1109) exhibited higher virulence than P. aeruginosa PA14 and the other clinical isolates tested in this study. Altogether, these findings demonstrated that P. aeruginosa ST463, with high pyocyanin production, is a high-risk clone in patients, suggesting that more attention should be paid to the control of the dissemination of such clones in the clinic.

CONCLUSION
The present study indicated that clinical P. aeruginosa poses a potential threat to human health because of the presence of multiple virulence factors and antibiotic resistance genes. The results suggested that the strain ST463 most likely emerged as a hypervirulent clone of P. aeruginosa as a result of a unique combination of pyocyanin production and virulence genes, including exoU + /exoS + , and pldA. Additionally, our study proves that the utility of genome sequencing in understanding and monitoring the epidemiology of clinically significant nosocomial clones, which will lead to improved control strategies. Nevertheless, the dissemination, evolution, and fitness cost of clone ST463 remain unclear.

DATA AVAILABILITY STATEMENT
Illumina sequences generated and used in this study are deposited and available at the NCBI website under BioProject ID: PRJNA716108 and part of PRJNA648026. All P. aeruginosa isolates (30)

ETHICS STATEMENT
The studies involving human participants were reviewed and Ethical approval was approved by the Ethics Committee of The