Anneloes van Veen | TU Delft Repository

Detection methods for carbapenem-resistant Pseudomonas aeruginosa in surface water and wastewater

Journal article (2025) - Selvi N. Shahab, Anneloes van Veen, Merel A. Kemper, Amber Rijfkogel, Margreet C. Vos, Anis Karuniawati, Juliëtte A. Severin, Heike Schmitt

Water systems can act as an important reservoir for Pseudomonas aeruginosa, which can pose public health risks during human contact. Carbapenem resistance is one of the most concerning resistances in P. aeruginosa making it a high-priority pathogen according to the World Health Organization (WHO), due to its ability to cause difficult-to-treat infections. Culture methods for detecting carbapenem-resistant P. aeruginosa (CRPA) have generally been developed for clinical samples but rarely for water. Therefore, the aim of this study was to develop a standardized and widely applicable method for detecting CRPA in water samples, both in surface water and wastewater. Growth of well-characterized strains of CRPA was determined in four different selective agar media which were each supplemented with imipenem, meropenem, or ceftazidime, including after an enrichment. Wastewater samples presumptively containing CRPA (naturally contaminated samples) and surface water samples spiked with well-characterized CRPA strains were filtered and cultured using selective agar plates supplemented with imipenem and meropenem, with and without a prior enrichment step. M-PA-C agar plates supplemented with imipenem performed best compared to other selective agar plates with respect to growth of well-characterized CRPA strains and suppression of background growth of other bacteria and carbapenem-susceptible P. aeruginosa (CSPA) with positive predictive value of 80–100 %. A prior step using asparagine proline broth with vancomycin is recommended for higher specificity considering direct plating was unsuccessful due to extensive background growth. A combination of nonselective enrichment with selective plating (i.e., addition of imipenem to the plate) is recommended to achieve low limits of detection. The addition of imipenem in the broth instead of in solid agar plates eliminated some of the CRPA strains, producing an increase in the lower limit of detection and a lower PPV (32 % vs 73 %). ...

Water systems can act as an important reservoir for Pseudomonas aeruginosa, which can pose public health risks during human contact. Carbapenem resistance is one of the most concerning resistances in P. aeruginosa making it a high-priority pathogen according to the World Health Organization (WHO), due to its ability to cause difficult-to-treat infections. Culture methods for detecting carbapenem-resistant P. aeruginosa (CRPA) have generally been developed for clinical samples but rarely for water. Therefore, the aim of this study was to develop a standardized and widely applicable method for detecting CRPA in water samples, both in surface water and wastewater. Growth of well-characterized strains of CRPA was determined in four different selective agar media which were each supplemented with imipenem, meropenem, or ceftazidime, including after an enrichment. Wastewater samples presumptively containing CRPA (naturally contaminated samples) and surface water samples spiked with well-characterized CRPA strains were filtered and cultured using selective agar plates supplemented with imipenem and meropenem, with and without a prior enrichment step. M-PA-C agar plates supplemented with imipenem performed best compared to other selective agar plates with respect to growth of well-characterized CRPA strains and suppression of background growth of other bacteria and carbapenem-susceptible P. aeruginosa (CSPA) with positive predictive value of 80–100 %. A prior step using asparagine proline broth with vancomycin is recommended for higher specificity considering direct plating was unsuccessful due to extensive background growth. A combination of nonselective enrichment with selective plating (i.e., addition of imipenem to the plate) is recommended to achieve low limits of detection. The addition of imipenem in the broth instead of in solid agar plates eliminated some of the CRPA strains, producing an increase in the lower limit of detection and a lower PPV (32 % vs 73 %).

Pseudomonas aeruginosa carriage and associated risk factors in healthy individuals and patients from Rotterdam, Rome, and Jakarta

Journal article (2025) - Anneloes van Veen, Selvi N. Shahab, Maurizio Sanguinetti, Anne F. Voor in ’t holt, Juliëtte A. Severin, Heike Schmitt, Amber Rijfkogel, Margreet C. Vos, Yulia Rosa Saharman, Anis Karuniawati, Silvia Zelli, Desy De Lorenzis, Giulia Menchinelli, De Angelis De Angelis

Pseudomonas aeruginosa may colonize humans, however, epidemiological data are scarce. Here, we determined overall and body site-specific carriage rates and associated risk factors among healthy individuals and newly admitted patients in three major cities. This cross-sectional study was conducted in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia) between 2022–2024. Adult healthy individuals and newly admitted patients were asked to provide throat, navel, and rectal/perianal swabs, and to complete a questionnaire. Univariable and multivariable analyses were performed to determine factors associated with P. aeruginosa carriage. Carriage rates differed significantly between cities (p < 0.001), and were lowest in Rome (healthy individuals 4.8%; patients 6.5%), followed by Rotterdam (healthy individuals 12.0%; patients 12.7%), and Jakarta (healthy individuals 28.6%; patients 24.0%). In carriers from Rotterdam, P. aeruginosa was most often detected in perianal swabs, while mostly in throat swabs among carriers from Rome and Jakarta. P. aeruginosa carriage had a seasonal association in patients from Rotterdam (p = 0.014) and Jakarta (p = 0.020). Among patients from Jakarta, female sex (aOR 1.98, 95% CI 1.02–3.84; p = 0.045) was associated with P. aeruginosa carriage. Overall, P. aeruginosa carriage rates and colonized body sites differ between cities and are likely associated with climate differences. Our findings warrant setting-specific adaptations of screening strategies and surveillance programs. ...

Sources and Transmission Routes of Carbapenem-Resistant Pseudomonas aeruginosa

Study Design and Methodology of the SAMPAN Study

Journal article (2025) - Anneloes van Veen, Selvi N. Shahab, Anne F. Voor in ’t holt, Silvia Zelli, Merel Kemper, Anniek E.E. de Jong, Sima Mohammadi, Heike Schmitt, Juliëtte A. Severin, More Authors...

Background/Objectives: The global spread of carbapenem-resistant Pseudomonas aeruginosa (CRPA) warrants collaborative action. Guidance should come from integrated One Health surveillance; however, a surveillance strategy is currently unavailable due to insufficient knowledge on the sources and transmission routes of CRPA. The aim of the SAMPAN study (“A Smart Surveillance Strategy for Carbapenem-resistant Pseudomonas aeruginosa”) is to develop a globally applicable surveillance strategy. Methods: First, an international cross-sectional study will be conducted to investigate CRPA in clinical and environmental settings in Rotterdam (The Netherlands), Rome (Italy), and Jakarta (Indonesia). Screening cultures and risk factor questionnaires will be taken from healthy individuals and patients upon hospital admission. Clinical CRPA isolates will also be included. Additionally, samples will be taken twice from wet hospital environments and monthly from the hospitals’ (drinking) water system, hospital and municipal wastewater treatment plants, and receiving rivers. Whole-genome sequencing will be performed to characterize CRPA isolates and determine the genetic relatedness among the isolates from different reservoirs. Findings from the cross-sectional study, combined with expert elicitation using a Delphi method, will serve as the input for the surveillance strategy. Conclusions: The SAMPAN study will provide a broader understanding of the sources and transmission routes of CRPA. Therewith, the development of a globally applicable smart surveillance strategy will be made possible, delivering information that is needed to guide actions against the spread of CRPA. ...