Evaluation of swabbing methods for culture and non-culture-based recovery of multidrug-resistant organisms from environmental surfaces

Abstract

Objectives: Sponge-Sticks (SS) and ESwabs are frequently utilized for detection of multidrug-resistant organisms (MDROs) in the environment. Head-to-head comparisons of SS and ESwabs across recovery endpoints are limited.

Design: We compared MDRO culture and non-culture-based recovery from (1) ESwabs, (2) cellulose-containing SS (CS), and (3) polyurethane-containing SS (PCS).

Methods: Known quantities of each MDRO were pipetted on a stainless-steel surface and swabbed by each method. Samples were processed, cultured, and underwent colony counting. DNA was extracted from sample eluates, quantified, and underwent metagenomic next-generation sequencing (mNGS). MDROs underwent whole genome sequencing (WGS). MDRO recovery from paired patient perirectal and PCS-collected environmental samples from clinical studies was determined.

Setting: Laboratory experiment, tertiary medical center, and long-term acute care facility.

Results: Culture-based recovery varied across MDRO taxa, it was highest for vancomycin-resistant Enterococcus and lowest for carbapenem-resistant Pseudomonas aeruginosa (CRPA). Culture-based recovery was significantly higher for SS compared to ESwabs except for CRPA, where all methods performed poorly. Nucleic acid recovery varied across methods and MDRO taxa. Integrated WGS and mNGS analysis resulted in successful detection of antimicrobial resistance genes, construction of high-quality metagenome-assembled genomes, and detection of MDRO genomes in environmental metagenomes across methods. In paired patient and environmental samples, multidrug-resistant Pseudomonas aeruginosa (MDRP) environmental recovery was notably poor (0/123), despite detection of MDRP in patient samples (20/123).

Conclusions: Our findings support the use of SS for the recovery of MDROs. Pitfalls of each method should be noted. Method selection should be driven by MDRO target and desired endpoint.

Figure 1.

Photographs illustrating methods to assess the effectiveness of surface sampling for culture and metagenomic analysis. (A) example of quadruplicate bacterial dilution plating to determine starting inoculum and post-swabbing colony recovery; (B) Different sampling methods assessed (from left to right) Ewab, cellulose containing sponge-sticks, polyurethane containing sponge-sticks (C) 8 inch by 12 inch stainless steel surfaces which bacterial suspension was applied to (D) MDRO colonies on selective and differential chromogenic agars (from right to left) salt-mannitol MRSA selective agar, VRE selective agar, ESBL selective agar, MacConkey agar.

Figure 2.

Recovery of percent of starting inoculum by culture (A), and nucleic acid concentration after extraction (B) for each MDRO category and sampling method. Abbreviations: CRAB: carbapenem-resistant Acinetobacter baumannii, CRPA: carbapenem-resistant Pseudomonas aeruginosa, CS: cellulose-containing Sponge-Stick, ESBL: extended spectrum beta-lactamase producing Enterobacterales, MRSA: Methicillin-resistant Staphylococcus aureus PCS: polyurethane-containing Sponge-Stick, VRE: vancomycin-resistant Enterococcus.

Figure 3.

Family-level taxonomic relative abundance of metagenomes from each MDRO category positive control and sponge combination pre-(A) and post- (B) bioinformatic decontamination. Decontaminated metagenomes all had 0.6 proportional abundance of positive control MDRO genomes but sponge sticks were closer to 1.0 relative abundance, likely due to higher biomass and DNA yields. (C) MDRO genome coverage depth and breadth within each sampling method metagenome sequenced with equal target depth, demonstrating consistent 100% coverage breadth across methods but lower depth with ESwabs. Abbreviations: CS: cellulose-containing Sponge-Stick, ESBL: extended spectrum beta-lactamase producing Enterobacterales, MRSA: Methicillin-resistant Staphylococcus aureus, PCS: polyurethane-containing Sponge-Stick, VRE: vancomycin-resistant Enterococcus.