Shahab, S. N. | bioRxiv Science

bioRxiv2024

Development of a highly-sensitive method to detect the carriage of carbapenem-resistant Pseudomonas aeruginosa in humans

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) causes severe and potentially life-threatening infections in hospitalized patients with mortality rates of more than 40%. To detect CRPA carriage in humans for surveillance purposes or to prevent spread and outbreaks in hospitals, a highly-sensitive culture method for CRPA carriage in humans is needed. We aimed to develop such a highly-sensitive method, that would be feasible in laboratories with limited resources. In this study, seven well-defined CRPA strains belonging to high-risk clones were used, including one CRPA without a carbapenemase gene and six carbapenem-resistant isolates with carbapenemase genes. We applied a stepwise approach wherein we included four enrichment broths and eight Pseudomonas aeruginosa-selective culture media. Spiking experiments were performed to further evaluate the combination of the most sensitive enrichment broths and selective agar plates in human samples. The two most sensitive enrichments broths were TSB-vancomycin and TSB-vancomycin with 2 mg/L imipenem and the most sensitive selective agar plates were Pseudomonas isolation agar Becton Dickinson, Pseudomonas isolation agar Sigma-Aldrich, and M-PA-C (Becton Dickinson). After the spiking experiment, the best method for detecting CRPA based on the sensitivity and the selectivity was the combination of TSB-vancomycin with 2 mg/L imipenem as an enrichment broth for overnight incubation, followed by subculturing the broth on M-PA-C agar plate. We have thus developed a highly-sensitive selective method to detect CRPA carriage in humans, which can also be applied in limited-resource laboratories. This may contribute to an overall effort to control CRPA.

microbiology↗

bioRxiv2024

Stability of four carbapenem antibiotics in discs used for antimicrobial susceptibility testing

In low-to middle-income countries, microbiological laboratories often use disc diffusion for antimicrobial susceptibility testing (AST). Reliable AST of carbapenem antibiotics is crucial for treatment decisions and surveillance purposes. Transport and storage conditions of materials used for AST are critical and may be challenging in some settings, where temperature cannot always be controlled. This study aimed to test the stability of four carbapenems in discs for AST under unfavourable conditions, i.e., at room temperature and 35{degrees}C for up to 72 hours. Imipenem, meropenem, ertapenem, and doripenem discs from three brands, Oxoid, Becton Dickinson, and HiMedia, containing 10 g of antibiotic were included. Discs were exposed to six unfavourable conditions and the recommended storage-condition as control. Subsequently, disc diffusion testing following the EUCAST guidelines was performed with four well-defined strains of Escherichia coli with different susceptibility profiles to carbapenems. The inhibition zone diameters were measured after 16-18 hours of incubation at 35{+/-}2{degrees}C. All experiments were executed in triplicate. In parallel, the carbapenems degradation was observed using a spectrophotometric method. Our study revealed that carbapenem discs were generally stable for AST although the concentration of most carbapenem antibiotics in discs decreased over time. Overall, imipenem (Oxoid and Becton Dickinson) discs were the most stable. Meropenem discs were less stable when exposed to 35{degrees}C than at room temperature. Concentrations of carbapenems in HiMedia discs were higher than those in Oxoid and Becton Dickinson. For carbapenem AST using disc diffusion in a rural area, we recommend using imipenem discs from Oxoid or Becton Dickinson. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/599257v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ca4d5aorg.highwire.dtl.DTLVardef@9c54bborg.highwire.dtl.DTLVardef@17ee220org.highwire.dtl.DTLVardef@12fcfc0_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIIn remote areas, transportation conditions of AST discs may be challenging C_LIO_LIDiscs with four carbapenems from three brands were tested using six conditions C_LIO_LICarbapenem discs were generally stable for AST C_LIO_LIIn unfavourable conditions, concentrations in the discs degraded over time C_LIO_LIImipenem (Oxoid and Becton Dickinson) discs were the most stable C_LI

microbiology↗