| Summary: | <i>Pseudomonas aeruginosa</i> is a leading cause of hospital-acquired infections worldwide. Biofilm production, antibiotic resistance, and a wide range of virulence factors contribute to their persistence in nosocomial environments. We describe an outbreak caused by a multidrug-resistant <i>P. aeruginosa</i> strain in an ICU. Antibiotic susceptibility was determined and <i>bla</i><sub>PER-1</sub> and <i>qnrVC</i> were amplified via PCR. Clonality was determined using PFGE and biofilm formation was studied with a static model. A combination of antibiotics was assessed on both planktonic cells and biofilms. WGS was performed on five isolates. All isolates were clonally related, resistant to ceftazidime, cefepime, amikacin, and ceftolozane-tazobactam, and harbored <i>bla</i><sub>PER-1</sub>; 11/19 possessed <i>qnrVC</i>. Meropenem and ciprofloxacin reduced the biofilm biomass; however, the response to antibiotic combinations with rifampicin was different between planktonic cells and biofilms. WGS revealed that the isolates belonged to ST309 and serotype O11. <i>bla</i><sub>PER-1</sub> and <i>qnrVC6</i> were associated with a tandem of IS<i>CR1</i> as part of a complex class one integron, with <i>aac(6′)-Il</i> and <i>ltrA</i> as gene cassettes. The structure was associated upstream and downstream with Tn<i>4662</i> and flanked by direct repeats, suggesting its horizontal mobilization capability as a composite transposon. ST309 is considered an emerging high-risk clone that should be monitored in the Americas.
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