Date of Award

January 2024

Document Type

Open Access Thesis

Degree Name

Master of Public Health (MPH)


School of Public Health

First Advisor

Windy Tanner

Second Advisor

Richard Martinello


Abstract Background: Environmental healthcare-associated pathogens are of particular public health concern, not only as potential contributors to the escalating rates of hospital-acquired infections1 noted during the SARS CoV-2 pandemic, but also as valuable indicators for multidrug resistance2. Their significance extends to infection prevention and control, emphasizing the pivotal role of shaping specific interventions within healthcare settings.

Rationale: The study investigates R2A agar’s efficacy in recovering environmental pathogens, specifically carbapenem-resistant Enterobacteriaceae (CRE), crucial for enhancing infection prevention and control in healthcare settings.

Objectives: In this prospective study conducted within hospital environments, our objective is to identify superior culture methods for recovering chlorine-injured organisms from sink drains. Through this study, we endeavor to enhance our understanding of pathogen recovery techniques, particularly in environments where pathogens are stressed and exposed to cleaning products. Effective recovery is crucial for comprehensive epidemiological analysis. Additionally, we aim to isolate, quantify, and evaluate the susceptibility of these recovered pathogens to carbapenem antibiotics.

Methods: In this prospective study, a variety of agars was systematically tested and supplementary laboratory techniques, including replica plating and culture, was employed in an effort to identify an optimal recovery medium. Through these tests, recovery of bacteria while minimizing the growth of non-target organisms was essential; thus facilitating more accurate and efficient research in the field of antibiotic resistance in environmental settings. Isolated colonies underwent further testing including Kirby Bauer susceptibility testing and MALDI-TOF mass spectrometry for bacterial identification. CLSI standards were utilized for validation of these results.

Results: Notable differences were observed in colony forming units (CFUs) between TSA and R2A media, with R2A consistently yielding higher recovery for CHROMagar and KPC (mean CFU counts of 236.62 and 193.46, respectively), compared to TSA. However, TSA showed a slightly higher mean CFU count for ESBL (55.54) than R2A. The mean zone of inhibition by colony type, measured in millimeters (mm) using Kirby-Bauer disc diffusion, varied among different colony types: teal (11.75), metallic blue (12.65), cream (13.88), white (0), purple (25.67), dark blue with red halo (17.19), yellow (6.75), red pink (15.25), green (2.33), and translucent (4.23). Antimicrobial susceptibility testing, coupled with MALDI-tof results, revealed susceptibility to Imipenem (IPM) (average zone of inhibition of approximately 28 mm) but resistance to Ertapenem (ETP) (average zone of inhibition of approximately 12 mm) among all Enterobacteriaceae isolates, consistent with CLSI standards. Distribution of bacterial isolates identified through MALDI-TOF mass spectrometry was analyzed, focusing on family categorization. A total of 70 isolates were examined (n=70). The most prevalent family observed was Pseudomonadaceae (30%, n=21). Notably, Enterobacteriaceae (15.7%, n=11) emerged as another significant family. This distinction underscores the prominence of Enterobacteriaceae within the Enterobacterales order and highlights the diverse array of bacterial families within our sample population. Such findings emphasize the critical importance of comprehensively understanding CRE-resistant bacteria. Furthermore, analysis of zones of inhibition with Ertapenem showed varied responses among colony types, with R2A medium demonstrating a higher yield of Enterobacteriaceae.

Conclusion: ​​ We investigated the efficacy of R2A agar in recovering environmental pathogens, particularly CRE. The addition of replica plating significantly improved both recovery rates and colony forming unit (CFU) counts. Combining R2A with replica plating emerged as the optimal approach for enhancing recovery efficiency. Antimicrobial susceptibility testing confirmed resistance to Ertapenem among all Enterobacteriaceae isolates, consistent with CLSI standards, highlighting the urgency of addressing CRE bacteria. Furthermore, MALDI-TOF mass spectrometry analysis revealed a diverse array of bacterial families within the sample population, with Pseudomonadaceae and Enterobacteriaceae emerging as significant families. This underscores the critical importance of comprehensively understanding CRE bacteria, especially amidst the context of hospital-acquired infections and rising rates of resistance. The findings provide valuable insights for shaping specific interventions within healthcare settings, emphasizing the pivotal role of effective microbiological culture methods for pathogen recovery and subsequent epidemiological analysis.


This is an Open Access Thesis.

Open Access

This Article is Open Access