Genomic Characterization of Mutli-Drug Resistant Pseudomonas aeruginosa Clinical Isolates: Evaluation and Determination of Ceftolozane/Tazobactam Activity and Resistance Mechanisms

Resistance to ceftolozane/tazobactam (C/T) in Pseudomonas aeruginosa is a health concern. In this study, we conducted a whole-genome-based molecular characterization to correlate resistance patterns and β-lactamases with C/T resistance among multi-drug resistant P. aeruginosa clinical isolates. Resistance profiles for 25 P. aeruginosa clinical isolates were examined using disk diffusion assay. Minimal inhibitory concentrations (MIC) for C/T were determined by broth microdilution. Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context. The clonal relatedness was evaluated using MLST, PFGE, and serotyping. All the isolates were resistant to C/T. At least two β-lactamases were detected in each with the bla OXA-4, bla OXA-10, bla OXA-50, and bla OXA-395 being the most common. bla IMP-15, bla NDM-1, or bla VIM-2, metallo-β-lactamases, were associated with C/T MIC >256 μg/mL. Eight AmpC variants were identified, and PDC-3 was the most common. We also determined the clonal relatedness of the isolates and showed that they grouped into 11 sequence types (STs) some corresponding to widespread clonal complexes (ST111, ST233, and ST357). C/T resistance was likely driven by the acquired OXA β-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, and metallo- β-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results revealed C/T resistance determinants and patterns in multi-drug resistant P. aeruginosa clinical isolates. Surveillance programs should be implemented and maintained to better track and define resistance mechanisms and how they accumulate and interact.


INTRODUCTION
Antimicrobial resistance has been a soaring global health care tolling problem (Tacconelli et al., 2018;Moghnieh et al., 2019). Pseudomonas aeruginosa is one of the leading multidrug resistant (MDR) nosocomial pathogens worldwide and defined by the World Health Organization as a critical health concern with limited effective treatment options and is associated with poor clinical outcomes (Weiner et al., 2016;Tacconelli et al., 2018). MDR P. aeruginosa isolates have a broad variety of mechanisms mediating antimicrobial resistance (Gellatly and Hancock, 2013;Loṕez-Causapéet al., 2018). These include the up-regulation of efflux pumps, loss of outer membrane porins, the production of AmpC, extended-spectrum b-lactamases (ESBLs) and carbapenemases, and modification of antimicrobial target sites (Lister et al., 2009;Zavascki et al., 2010;Bassetti et al., 2019). The overproduction of AmpC b-lactamase was also linked to cephalosporins resistance and which was not reversed by blactamase inhibitors such as tazobactam and clavulanic acid (Sligl et al., 2015).
Two drug combinations were developed to treat infections caused by resistant Gram-negative bacteria, namely ceftazidime/ avibactam and ceftolozane/tazobactam (C/T) (Sold under the brand name Zerbaxa) (Wright et al., 2017). Ceftolozane is a cephalosporin derivative of ceftazidime with an intrinsic broad activity and is not hydrolyzed by most broad-spectrum blactamases such as ESBLs and AmpCs (van Duin and Bonomo, 2016). Ceftolozane is particularly active against P. aeruginosa exhibiting AmpC efflux pumps overexpression (Moyáet al., 2012;Giacobbe et al., 2018), and has a heavier pyrazole substituent at the 3-position side chain instead of the lighter pyridium in ceftazidime, enhancing steric hindrance and interfering with AmpC hydrolytic activity (van Duin and Bonomo, 2016). The combination of ceftolozane and the blactamase inhibitor tazobactam proved to be active against many, but not all, ESBL-producing Gram-negative bacteria (Farrell et al., 2014). In particular, C/T combination was active against MDR and carbapenem-resistant P. aeruginosa (Giacobbe et al., 2018). The overall clinical success rate was reported to be 76.2% among MDR and extensively drug-resistant (XDR) P. aeruginosa (Maraolo et al., 2020). C/T drug combination was also more effective compared to polymyxins or aminoglycosides (Pogue et al., 2019). However, regional variations in C/T resistance within MDR P. aeruginosa were reported (Farrell et al., 2014;Sid Ahmed et al., 2019;O'Neall et al., 2020), and AmpC hyperproduction was a factor linked to C/T resistance in P. aeruginosa (Cabot et al., 2014). C/T was also less efficient against ESBL producing Escherichia coli and P. aeruginosa (Ortiz de la Rosa et al., 2019). The C/T activity against the Gram-negative ESBL producers, including E. coli, Klebsiella pneumoniae, and P. aeruginosa, recovered from clinical settings in Lebanon was recently tested by Araj et al. (2020), and which revealed that the C/T against MDR E. coli and K. pneumoniae isolates were much lower (Araj et al., 2020). In light of the rapid increase in the number of C/T P. aeruginosa resistant isolates we thought of studying C/T activity against MDR P. aeruginosa, determining the susceptibilities of the isolates, and conducting a genomebased molecular characterization.

Ethical Approval
Ethical approval was not required. The isolates were collected as part of routine clinical care and patient data collection. No additional isolates were collected beyond those obtained from routine clinical care, and no diagnostic or treatment decisions were affected by the outcomes of this study.

Bacterial Isolates and Identification
A total of 25 P. aeruginosa isolates were collected and identified by the Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) system (Bruker Daltonik, GmbH, Bremen, Germany) at the clinical microbiology laboratory of the American University of Beirut Medical Center (AUBMC), Beirut, Lebanon between December 2017 and November 2018. AUBMC is around 350-bed tertiary care major hospital in the country. The isolates were designated as ZBX-P1 to ZBX-P25.

Whole-Genome Sequencing
DNA extraction was performed using the NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany) according to the manufacturer's instructions followed by long-read sequencing of the isolates (ZBX-1 to ZBX-25). PacBio long-read sequencing on the Sequel I platform (Pacific Biosciences, CA, USA) was performed. Library preparation was according to the manufacturer's instructions for microbial isolate multiplexing. G-tubes (Covaris, USA) were used for DNA shearing, and no size selection was performed. Resulting contigs were polished with Pilon, v1.23 (Walker et al., 2014), and the overlapping ends of chromosomes were trimmed after manual inspection of reads mapped by BWA-MEM algorithm as implemented in BWA, v0.7.17 (Li and Durbin, 2010), and Bowtie, v2.3.4.2 (Langmead and Salzberg, 2012). Genome assembly was done using HGAP4 with minimum seed coverage of 30x (Chin et al., 2013).

Data Availability
The Whole Genome Shotgun project was deposited at DDBJ/ ENA/GenBank under the accession numbers presented in S1 Table.

Resistance Genomics
Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context ( Figure 3 and Table 1). Between two to four b-lactamases were detected in the sequenced genomes; all were chromosomal except for bla   (Table 1). bla IMP-15 , bla NDM-1 , or bla VIM-2 , metallo-b-lactamases, were associated with C/T MIC >256 mg/mL. Other b-lactamases were also detected with the most common being bla OXA-395 , bla OXA-50 , bla OXA-4 , and bla OXA-10 , respectively (Figure 3). MIC distributions are shown in (Figure 2). All C/T resistant isolates with MIC > 256 were either intermediate (n =1) or resistant to ceftazidime (n = 15). Of these, nine had four b-lactamases with one being a metallo-b-lactamase.
Finally, we compared 27 porin encoding genes with that of P. aeruginosa PAO1. The highest variability was observed in oprD, opdD (PA4501 and PA1025), and oprQ. Deletions and premature stop codons (truncations) were detected throughout FIGURE 2 | PFGE dendrogram and Serotype in the C/T P. aeruginosa isolates. Dendogram was generated by BioNumerics software version 7.6.1 showing the relationship of the isolates based on their banding patterns generated by SpeI restriction digestion. Isolates' STs, and pulsotypes (PT) are shown.

DISCUSSION
Choosing the appropriate antimicrobial agent to treat infections caused by resistant bacteria would significantly decrease infection-linked morbidity and mortality. C/T has increased activity against resistant P. aeruginosa isolates and is an important treatment option in institutions with high rates of pseudomonal infections (Humphries et al., 2017;Hirsch et al., 2020). The emergence of C/T resistance in MDR/XDR P. aeruginosa isolates, resistant to most b-lactams and having several resistance mechanisms, is very likely to happen. In this study, we used long-read whole-genome sequencing approach to study the resistance genomics and clonal relatedness of 25 C/T resistant P. aeruginosa. The isolates belonged to 11 STs and 23 pulsotypes, exhibited C/T MIC of 1.5 to > 256 mg/mL, showed resistance to the other tested b-lactams including ceftazidime (88%; n=22), ciprofloxacin (68%; n=17), and imipenem (68%; n=17). Moreover, isolates had two to four b-lactamases and 10 were positive for bla IMP-15 , bla NDM-1, or bla VIM-2 , and were intrinsic AmpC producers.
PDC variants in this study were assigned according to substitutions in AmpC described by Rodrıǵuez-Martıńez et al. (2009) and accordingly we identified eight ampC variants, PDC-3 being the most common. T79A (T105A non-processed peptide) detected in PDC-3 (Rodrıǵuez-Martıńez et al., 2009), was previously found to be prevalent in carbapenem-resistant clinical isolates. Berrazeg et al. (2015), however, overexpressed PDC-3 (T79A) in porin OprD-negative strain to test if it would potentiate the AmpC action and revealed that T79A variation doesn't broaden the substrate spectrum of AmpC (Berrazeg et al., 2015). They concluded that PDC common polymorphisms had negligible impact on AmpC activity while confirming that mutations occurring in specific regions of the substratebinding pocket could enhance the catalytic efficiencies and as a result increase the hydrolytic activity of P. aeruginosa AmpC on cephalosporins including ceftolozane. On the other hand, Fernańdez-Esgueva et al. (2020) showed that C/T resistance was associated with AmpC mutations including a novel one (PDC-388; G183V) (Fernańdez-Esgueva et al., 2020) and in line with this Fraile-Ribot et al. (2018) also cloned AmpC variants 222,and 223) in ampC-deficient derivative of PAO1 (Fraile-Ribot et al., 2018). The cloned AmpC variants showed increased ceftolozane/tazobactam and ceftazidime/avibactam MICs compared with wild type AmpC with the associated polymorphisms being located within the Ω loop and selected FIGURE 3 | Detailed phenotypic and genotypic resistance information of the C/T resistant P. aeruginosa isolates. ST, Sequence type. Classes of antibiotic resistance genes are marked as follows: aminoglycoside, phenicol, macrolide, tetracycline, fosfomycin, quinolone, rifampicin, trimethoprim, and sulphonamide resistance genes. MIC, Minimum inhibitory concentration (µg/ml), DD, Diffusion diameter (mm). Black, present. in vitro upon C/T exposure (Cabot et al., 2014;Haidar et al., 2017). We detected the characteristic OprD mutation (Q142X) associated with C/T resistance (Fraile-Ribot et al., 2018) in three of the study isolates, ZBX-P12, ZBX-P16, and ZBX-P23 (C/T MIC > 256 mg/mL). Moreover, C/T resistance emergence was previously reported in isolates producing horizontally acquired b-lactamases such as OXA-10 and OXA-2 (Fraile- Ribot et al., 2018), and ESBLs (Ortiz de la Rosa et al., 2019). In general, C/T may be ineffective against isolates carrying carbapenemases including class A and class D (OXA) b-lactamases and it's inactive against metallo-blactamases (Hirsch et al., 2020;Karlowsky et al., 2021). Extended-spectrum OXAs were also noted as infrequent cause of ceftolozane resistance (Livermore et al., 2009;Juan et al., 2010). Our results mainly agreed with C/T resistance being linked to horizontally acquired b-lactamases. We detected two to four b-lactamases in the sequenced genomes. In line with this, Sid Ahmad et al. (2019) reported a strong association between MDR P. aeruginosa isolates displaying MIC 256 to C/T and the presence of OXA-10, VIM-2, and OXA-488 with the highest association frequency being with class C and D b-lactamases. We also had metallo-b-lactamases including bla IMP-15 , bla NDM-1, or bla VIM-2 and which were invariably (except for one isolate) linked to C/T MIC >256 mg/mL.
Infections caused by MDR P. aeruginosa could be treated with C/T, clinical studies evaluating optimal dosing and using combined therapy are recommended (Fraile-Ribot et al., 2018). Mutations occurring within the substrate-binding pocket could increase the hydrolytic activity of P. aeruginosa AmpC on cephalosporins including ceftolozane. Horizontally acquired blactamases, and intrinsic AmpC modifications are the main mechanisms leading to C/T resistance in MDR P. aeruginosa. Our data don't support the accumulation of mutations leading to the overexpression or structural modification of AmpC and so it's more likely that C/T resistance was driven by the acquired OXA b-lactamases such as OXA-10, OXA-50, ESBLs GES-1, GES-15 and VEB-1, and metallo-b-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results highlight the need to maintain active surveillance programs to better track and define resistance mechanisms and how they accumulate and interact.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.