Discussion

The present study is the first and the largest from the Middle East to address systematic surveillance, molecular epidemiology, microbiological and clinical characteristics, as well as clinical outcomes associated with infections secondary to MDR P. aeruginosa in a major hospital system. During the 3-year study period in Qatar, the overall prevalence of MDR P. aeruginosa was 5.6%, which is lower than reports from neighboring countries or regions. ^{Reference Al-Orphaly, Hadi and Eltayeb11} The prevalence of MDR P. aeruginosa initially peaked at 8.1% in the first year of the study, then decreased throughout the study until it reached 4.8% in the third year. At the start the of the study, the upward trend in the epidemiology of GNB, including MDR P. aeruginosa, prompted the monitoring process. The significant decline in the prevalence of MDR P. aeruginosa over the study period might be attributed to the efficacy of the antimicrobial stewardship program (ASP), which targeted broad-spectrum antimicrobials, including antipseudomonal agents. This effort began around 2015 across all hospitals in Qatar to curb rising AMR rates. ^{Reference Sid Ahmed, Abdel Hadi and Abu Jarir23} The downward trend of MDR P. aeruginosa demonstrates a probable positive impact of the ASP, supported by the observation that the overall proportion of clinical isolates of MDR P. aeruginosa did not change over the study period. In addition, we detected a substantial decrease in the use of antipseudomonal agents, reaching 30%, with no major changes in infection control and prevention practices (Table 1).

Similarly, a Cochrane Database systemic review demonstrated the success of ASP interventions to improve antibiotic prescribing in hospital inpatients and reduce AMR. ^{Reference Pakyz, Oinonen and Polk24} Our findings agree with those from another study from the United States that investigated the relationship of carbapenem restriction in 22 university teaching hospitals and the incidence of carbapenem-resistant P. aeruginosa. ^{Reference Defez, Fabbro-Peray and Bouziges25}

Regarding the isolation of MDR P. aeruginosa from different body sites, the distribution and rank order of P. aeruginosa isolates were generally in agreement with isolates obtained through the Global SENTRY Antimicrobial Surveillance Program 1997–1999. Respiratory isolates were most prevalent, followed by skin and soft-tissue, urine, and blood isolates. ^{Reference Gales, Jones, Turnidge, Rennie and Ramphal26} The low proportion of MDR P. aeruginosa bloodstream isolates in this study, which belonged to 13 different sequence types, suggests that cross transmission is not a major problem in the context of MDR P. aeruginosa bacteremia in Qatar. Furthermore, 95% of MDR P. aeruginosa infections were hospital acquired, and the leading risk factors for acquisition of MDR P. aeruginosa included a prior encounter with healthcare settings, colonization with a susceptible P. aeruginosa strain, as well as exposure to antimicrobials in the preceding 90 days. These findings are in line with those of previous studies. ^{Reference Ahmed, Abu Jarir and Abdel Hadid14}

Risk factors for acquisition and clinical outcomes for MDR P. aeruginosa were not related to age or sex, whereas the 30-day mortality rate significantly increased from 3.9% in the first year to 11.2% in the third year. This rate was significantly higher for critical care patients, and 31.3% of these cases were bloodstream infections (Table 2). This finding is in accordance with previous studies. ^{Reference Wisplinghoff, Bischoff, Tallent, Seifert, Wenzel and Edmond27,Reference Parkins, Gregson, Pitout, Ross and Laupland28} A noticeable feature of this study was the associated comorbidities with MDR P. aeruginosa acquisition (ie, diabetes mellitus, end-stage renal disease, malignancy, heart failure, and chronic lung diseases); however, this observational correlation probably stems from the association of comorbidities with prolonged hospital stay and infection acquisition. Such associations have been observed in other AMR morbidity and mortality studies. ^{Reference Defez, Fabbro-Peray and Bouziges25,Reference Micek, Wunderink and Kollef29}

Genomic analysis of major clones revealed the dominance of 5 major epidemic sequence types: ST235, ST357, ST389, ST1284, and ST233 (Table 4). These notorious epidemic clones exhibit high MIC values for ceftazidime-avibactam and ceftolozane-tazobactam, and they are responsible for spreading resistance globally, including in the Middle East. ^{Reference Perez, Hujer and Marshall30–Reference Abdouchakour, Aujoulat and Licznar-Fajardo32} Interestingly, most of ST235 and ST308 isolates were isolated from UTIs from patients who attended the outpatient department of the same hospital at HGH, and most ST357 isolates were obtained from patients the inpatient department of the same facility, which supports clonality of HAIs. On the other hand, all ST389 isolates (all from cystic fibrosis patients) and ST274 isolates were obtained from patients with respiratory tract infections, whereas most ST233 isolates were obtained from patients with bloodstream infections. These observations suggest that specific virulence factors are associated with certain sequence types that are site specific. ^{Reference Behzadi, Baráth and Gajdács33,Reference Francis, Stevenson and Porter34} The major clones, dominated by ST235 and ST357, have been associated with global epidemic dissemination and spread of MDR strains. These epidemic clones follow regional locations established at specific healthcare settings, which supports environmental endemicity and clonality of MDR P. aeruginosa (Table 4). ^{Reference Warnes, Bolker and Bonebakker22} Other less frequently isolated clones, such as ST389, ST1284, and ST233, seem to be endemic to our setting. Reports of ST1284 and ST233 are rare and seem to be highly resistant, harboring carbapenem resistance genes such as bla _VIM, which has also been observed in Brazil and Japan. ^{Reference Balero de Paula, Cayô and Streling35,Reference del Barrio-Tofiño, López-Causapé and Oliver36} In contrast, none of the previously reported high-risk P. aeruginosa clones, such as ST111 and ST175, were detected in the present study. ^{Reference Horcajada, Montero and Oliver9}

Our analysis of microbiological characteristics demonstrated a significant resistance profile, reaching or exceeding 90% for primary antipseudomonal agents (Table 3). This observation is worrying because it establishes that AMR for MDR P. aeruginosa is a significant challenge with limited treatment options, particularly because other available agents, such as colistin, demonstrated the highest susceptibility rates (97.5%) at the expense of associated adverse events. ^{Reference Spapen, Jacobs, Van Gorp, Troubleyn and Honoré37} Probably because of significant underlying embedded regional resistance profiles, even before the introduction of novel agents such as ceftazidime-avibactam and ceftolozane-tazobactam in our clinical practice, observed susceptibility of the collection for both agents was lower than other regions at 68.8% and 62.9%, respectively. ^{Reference Sid Ahmed, Abdel Hadi and Hassan38} In our previous study, we examined β-lactamase–mediated genes through selective genomic analysis of 75 MDR P. aeruginosa isolates, which included 42 isolates that were resistant to 1 or both agents. They all possessed class C and/or class D β-lactamases; 50% of isolates possessed class A β-lactamases; and 26.7% possessed class B β-lactamases. ^{Reference Sid Ahmed, Khan and Sultan39,Reference Sid Ahmed, Khan, Hadi, Skariah, Sultan, Salam, Al Khal, Söderquist, Ibrahim, Omrani and Jass40}

Additionally, genomic analyses of virulence factors in previous studies have detected the presence of mutations in distinct genes for MDR P. aeruginosa–encoded type III exotoxins: exoS, exoT, exoU, exoY, which are known to be associated with severe infections. ^{Reference Sato, Frank and Hillard13} In this study, comparative analysis of the presence or absence of P. aeruginosa virulence factors against sequence types, site of isolation, antipseudomonal agents, and clinical outcomes revealed that they are clustered within certain sequence types and are related to negative clinical outcomes, such as bloodstream infections, but are probably not related to AMR (Fig. 3).

In conclusion, our 3-year study of 525 clinical isolates of MDR P. aeruginosa is one of the largest global collections that established the initial relatively higher prevalence of resistant clinical isolates demonstrating significant AMR in Qatar, which was later ameliorated through effective surveillance, monitoring, and implementation of a successful ASP. We also observed the role of specific endemic clones that were geographically located demonstrating high-level resistance as well as being associated with specific infections such as bloodstream and exacerbation of infections in patients with cystic fibrosis probably related to certain underlying virulence factors. In the fight to control HAIs, we advocate continuous surveillance, monitoring as well as genomic analysis to understand pathogens epidemiology, microbiological characteristics as well as underlying mechanisms of virulence and resistance.