Background Information

Carbapenem-resistant Pseudomonas aeruginosa stands out as a major contributor to hospital-acquired infections, emerging as a global health priority. The World Health Organization (WHO) has designated multidrug-resistant (MDR) bacteria, including P. aeruginosa, Acinetobacter baumannii, and Enterobacterales, as critical priorities due to their resistance to crucial antibiotics. This resistance, often multifactorial, involves mechanisms such as efflux pumps, porin gene deficiencies, and cephalosporinase gene overexpression.

Carbapenem resistance, frequently linked to carbapenemase production like serine β-lactamases and metallo-β-lactamases (MβLs), aligns with high-risk global clones such as ST235, ST111, and ST277. The Brazilian endemic clone ST277, prevalent in the country, associates with carbapenem resistance due to MβL SPM-1 production. SPM-1-producing P. aeruginosa has been found in hospital sewage and wastewater treatment plants, suggesting potential dissemination in aquatic environments.

Despite genomic studies on human SPM-1-positive isolates, there is a lack of sequence data for environmental isolates. Recognizing the critical importance of such pathogens, this study aims to furnish genomic data and assess the virulence potential of carbapenem-resistant SPM-1-positive P. aeruginosa strains isolated from polluted urban rivers in Brazil.

The SPM-1 Pseudomonas aeruginosa strains that were used during this study were;

• Pa19 Tietê River, GenBank accession number: PHST01000000 and
• Pa151 (Pinheiros River, GenBank accession number: PHSS00000000.1.

They compared the sequences of these strains against publicly available genome sequences of clinical SPM-1 producing P. aeruginosa;

• CCBH4851 (catheter tip, GenBank accession number CP021380.2)
• PA1088 (urine, GenBank accession number CP015001.1)
• PA11803 (bloodstream, GenBank accession number: CP015003.1)
• PA12117 (bloodstream, GenBank accession number: LVXB00000000.1) and
• PA7790 (tracheal aspirate, GenBank accession number: CP014999.1)

Strains were retrieved from NCBI GenBank database.14 For SNP-based analysis (Single Nucleotide Polymorphisms), the genome of the P. aeruginosa strain PAO1 (ST549) was used as reference (GenBank accession number: AE004091.2)

Virulence potential of P. aeruginosa Pa19 and Pa151 strains was evaluated using the Galleria mellonella infection model

Whole Genome Sequencing and Genomic Analysis

1. Genomic DNA of Pa19 & Pa151 were extracted using PureLink Quick Gel Extraction & PCR Purifcation Combo Kit
2. Illumina paired-end libraries were constructed using a Nextera XT DNA Library Preparation Kit (Illumina Inc.), according to the manufacturer’s guidelines.
3. Whole genome sequencing was performed using an Illumina MiSeq platform with 300-bp read lengths.
4. Reads were de novo assembled using SPAdes 3.13,1 and the resulting contigs were automatically annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 3.2
5. Antibiotic resistance genes were predicted using ResFinder 4.13 and the Comprehensive Antibiotic Resistance Database (CARD)
6. Multi-locus Sequence Typing prediction was performed using MLST v.2.0.
7. Heavy metal (HM) resistance genes were manually identifed using the NCBI database6 and Geneious Prime version 2020.04
8. Phage prediction was performed by Genome Detective Virus Tool software
9. The rmtD gene was detected and aligned by BLASTn against the rmtD1 allele of the P. aeruginosa (PA0905 strain), recovered from a human patient (GenBank accession number. DQ914960).
10. virulence genes, effux systems, and regulators were determined through the Virulence Factor Database.8
11. Serotype was predicted using Past 1.0
12. SNP-based phylogenetic analysis was performed by using Prokka 1.13.4
13. Pangenome annotation, followed by Roary 3.13.0
14. RAxML-NG version 0.9.013 for phylogenetic construction and a maximum likelihood tree based on SNP alignment
15. Comparative genomic analysis was performed by BRIG v.0.95