2.1 Outbreak description and bacterial isolates
Between October 2013 and December 2014, a nosocomial outbreak caused byP. aeruginosa showing an MDR phenotype was detected in the Hematology ward of a Spanish tertiary hospital (Hospital General Universitario Gregorio Marañon -HGUGM-, Madrid, Spain –1350 beds-) (Acosta et al., 2020). Patients hospitalized at this ward showed an increased incidence of P. aeruginosa bacteremia, while this was not reflected at the overall hospital level. During this 15-month period, at least one isolate ofP. aeruginosa showing the same MDR phenotype was detected in 14 patients, while this microorganism was not found in the same ward during the previous 8 months.
As a first approach, PFGE was performed on 23 P. aeruginosa isolates: 12 available isolates out of the 14 outbreak suspected strains from 2013 and 2014, two of them sourcing from the same patient; 5 controls strains related to the hematology ward and 6 unrelated strains from the same time period. Moreover, a reference P. aeruginosa strain (ATCC 27853) was also included in the analysis in duplicate (Acosta et al., 2020). The methodology applied for PFGE has been described before (Sanchez-Carrillo et al., 2009).
The strains that grouped in the outbreak-pulsotype (P1) were further characterized by WGS (Table S1). This methodology differentiated three clusters within pulsotype P1: Group 1, which contained the P. aeruginosa strains correlated to the outbreak, and Groups 2 and 3, where isolates detected during the same period as the outbreak strains but with enough single nucleotide polymorphism (SNPs) to be considered as non-outbreak strains clustered -Figure 1- (Acosta et al., 2020).
With the outbreak-specific single nucleotide polymorphisms (SNPs) detected by WGS analysis, a multiplex allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) was designed for the rapid differentiation of the outbreak-related P. aeruginosa isolates (Group 1). The ASO-PCR was tested with a collection of isolates (n=32) from a broader period of time (2010-2018), allowing for the detection of new outbreak-related strains which were not considered part of the outbreak initially (Figure S1) (Acosta et al., 2020).
A total of n=67 available P. aeruginosa strains from this outbreak were included in this study (Table S1): 35 P. aeruginosaisolates analyzed by WGS (27 outbreak strains–all of them ST175–, and 8 control strains) and 32 isolates (16 outbreak and 16 non-outbreak strains) analyzed by ASO-PCR. All strains sourced from inpatients from HGUGM and were kept frozen at -80°C until further analysis. Isolates were cultured in 5% Columbia sheep blood agar plates at 37°C, and analyzed after a 24-hour overnight incubation period.