Four multidrug-resistant Pseudomonas aeruginosa isolates were cultured from intensive care unit wastewater. All isolates exhibited resistance to carbapenem and extended-spectrum beta-lactam antibiotics.
ABSTRACT
Four multidrug-resistant Pseudomonas aeruginosa isolates were cultured from intensive care unit wastewater. All isolates exhibited resistance to carbapenem and extended-spectrum beta-lactam antibiotics. Genome characterization revealed the presence of beta-lactamase resistance genes (blaPAO and blaOXA), and three out of the four isolates carried the blaNDM-1 gene encoding resistance against carbapenems.
ANNOUNCEMENT
Clinical wastewater is a source of pharmaceutical by-products, pathogens, and antibiotic-resistant bacteria (1–3), and may facilitate the spread of antimicrobial resistance in receiving waters if not treated. Four multidrug-resistant Pseudomonas aeruginosa isolates (WPB098, WPB099, WPB100, and WPB101) were cultured from wastewater onto selective chromogenic media, CHROMagar Klebsiella pneumoniae carbapenemase (KPC) agar (CHROMagar, Paris, France) from hospital wastewater samples of a clinical isolation ward in Singapore as described in Haller et al. (2017) (2). Antimicrobial susceptibility profiles of the isolates were evaluated using the AST-GN79 cards on the Vitek 2 compact system (bioMérieux, Marcy L'Étoile, France) according to the manufacturer’s instructions. All isolates showed resistance to 16 of the antibiotics tested, amikacin (AMK), ampicillin (AMP), ampicillin-sulbactam (SAM), cefazolin (CFZ), cefepime (FEP), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), ciprofloxacin (CIP), ertapenem (ETP), meropenem (MEM), gentamicin (GEN), nitrofurantoin (NIT), piperacillin-tazobactam (TZP), tobramycin (TOB), and trimethoprim-sulfamethoxazole (SXT). These results indicated that the isolated strains exhibit resistance to carbapenems and extended-spectrum beta-lactamase (ESBL) antibiotics.
For whole-genome sequencing, single colonies were picked from overnight growth and cultured in Luria Bertani broth overnight at 37°C. Total genomic DNA was extracted using the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA). Using the standard protocol, indexed sequencing libraries were prepared using a New England Biolabs Next Ultra DNA library prep kit for Illumina (New England BioLabs [NEB], Inc., MA, USA) and sequenced as paired-end reads (2 × 151 bp) using the Illumina HiSeq 4000 platform. A total of 26.1 million paired-end reads were obtained from four samples. Quality controlled reads with Phred scores of >20 were retained and assembled de novo using VelvetOptimiser 2.2.4 (4) with a minimum contig cutoff of 500 bp. Scaffolding was performed using Opera 1.4.1 (5) and finished using FinIS 0.3 with default settings (6). The genomes ranged between 6.5 and 6.9 Mbp and contained 80 to 98 contigs, with a G+C content of 66%. The assembled genomes had N50 values of between 234,330 and 312,695.
To identify antibiotic-resistant genes (ARGs) carried by each P. aeruginosa isolate, the assembled genomes were searched against the Resfinder 3.0 database using default parameters (7). All 4 isolates had ARGs that confer resistance to aminoglycoside [aph(3′)-IIb], beta-lactam (blaPAO and blaOXA), fosfomycin (fosA), and phenicol (catB7) antibiotics, while isolates WPB099, WPB100, and WPB101 encoded additional ARGs resistant to fluoroquinolone (crpP and qnrVC1), macrolide-lincosamide-streptogramin B (msrE), sulfonamide (sul2), and trimethoprim (dfrB5) antibiotics. The blaNDM-1 gene, which encodes the New Delhi metallo-beta-lactamase enzyme capable of hydrolyzing ESBL and carbapenem antibiotics, was detected in genomes of isolates WPB099, WPB100, and WPB101.
Data availability.
Whole-genome sequences of P. aeruginosa strains WPB098 to WPB101 were deposited in GenBank under accession numbers CP031876 to CP031879 (BioProject number PRJNA486613). Raw reads can be found in the NCBI Sequence Read Archive (SRA) under accession numbers SRR8002634 to SRR8002637.
ACKNOWLEDGMENTS
This work was funded by the Ministry of Education (MOE), Academic Research Fund (AcRF) Tier 2 grant (reference: MOE2015-T2-2-130).
We thank Koh Tse Hsien from the Department of Pathology at Singapore General Hospital (SGH) for supporting this research, and Paola Florez De Sessions for use of the GERMS platform.
REFERENCES
- 1.Le TH, Ng C, Chen H, Yi XZ, Koh TH, Barkham TMS, Zhou Z, Gin KYH. 2016. Occurrences and characterization of antibiotic resistant bacteria and genetic determinants of hospital wastewaters in a tropical country. Antimicrob Agents Chemother 62:7449–7456. doi: 10.1128/AAC.01556-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Haller L, Chen H, Ng C, Le TH, Koh HS, Barkham T, Sobsey M, Gin KYH. 2018. Occurrence and characteristics of extended-spectrum β-lactamase- and carbapenemase-producing bacteria from hospital effluents in Singapore. Sci Total Environ 615:1119–1125. doi: 10.1016/j.scitotenv.2017.09.217. [DOI] [PubMed] [Google Scholar]
- 3.Ng C, Tay M, Tan B, Le T-H, Haller L, Chen H, Koh TS, Barkham TMS, Thompson JR, Gin KYH. 2017. Characterization of metagenomes in urban aquatic compartments reveals high prevalence of clinically relevant antibiotic resistance genes in wastewaters. Front Microbiol 8:2200. doi: 10.3389/fmicb.2017.02200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Zerbino DR. 2011. Using the Velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinformatics Chapter 11:Unit 11.5. doi: 10.1002/0471250953.bi1105s31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Gao S, Sung WK, Nagarajan N. 2011. Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences. J Comput Biol 18:1681–1691. doi: 10.1089/cmb.2011.0170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Gao S, Bertrand D, Nagarajan N. 2012. FinIS: improved in silico finishing using an exact quadratic programming formulation, p 314–325. In Raphael B, Tang J (ed), Algorithms in bioinformatics, vol 7534 Springer, Berlin, Germany. doi: 10.1007/978-3-642-33122-0_25. [DOI] [Google Scholar]
- 7.Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
Whole-genome sequences of P. aeruginosa strains WPB098 to WPB101 were deposited in GenBank under accession numbers CP031876 to CP031879 (BioProject number PRJNA486613). Raw reads can be found in the NCBI Sequence Read Archive (SRA) under accession numbers SRR8002634 to SRR8002637.