Abstract
Purpose:
The SuperCP medium containing meropenem (2 mg/L) was evaluated for screening of carbapenem-resistant Pseudomonas species.
Materials and Methods:
It was evaluated using 29 meropenem-susceptible and 56 meropenem-nonsusceptible Pseudomonas-like clinical isolates, the latter exhibiting a variety of carbapenem-resistance mechanisms.
Results:
Its sensitivity and specificity of detection were found to be 91% and 100%, respectively. By testing spiked stools, an excellent performance of the medium was also observed for detection of carbapenem-resistant Pseudomonas aeruginosa, with a lowest detection limit ranging from 100 to 102 CFU/mL.
Conclusion:
This screening medium provides the opportunity to select carbapenem-resistant Pseudomonas and Pseudomonas-related isolates regardless of their resistance mechanism.
Introduction
Carbapenem-resistant Pseudomonas aeruginosa (CR-PA) have been recently recognized as an urgent and serious threat by the CDC and as priority pathogens by the WHO. They are sources of hospital-acquired infections causing significant mortality and morbidity in high risk-patient populations, and also of community-acquired pneumonia in cystic fibrosis patients. In addition, numerous outbreaks of CR-PA have been reported in particular in intensive care units worldwide. Several evidences suggest that patients infected with CR-PA have increased morbidity and mortality rates compared with those infected with meropenem-susceptible isolates. To address this clinical concern, ongoing surveillance of carbapenem resistance in P. aeruginosa is needed.
Although carbapenem resistance in P. aeruginosa is often related to permeability defects (in particular through OprD porin defects) that accounts for 10–15% of all P. aeruginosa clinical strains worldwide,1,2 acquisition of carbapenemase enzymes also represents a significant concern. An increasing number of carbapenemases belonging to different β-lactamase classes have been identified in that species. Reported β-lactamases in P. aeruginosa are of Ambler class A (KPC, GES derivatives), class B (NDM, VIM, IMP, GIM, DIM), and very rarely class D (OXA-40, OXA-181, OXA-198). 3
Whereas several screening media have been developed for screening carbapenem-resistant Enterobacterales and Acinetobacter baumannii (ChromID Carba, CHROMagar-Acinetobacter MDR medium, and SuperCarba medium), to the best of our knowledge, no screening medium has been developed for screening carbapenem-resistant Pseudomonas spp. and Pseudomonales-like species. Streaking for isolation of CR-PA on agar plates with meropenem disks has just been proposed by Goodmann et al. 4 when searching for CR-PA from stools, which does not correspond to an optimal screening procedure for detecting low quantity of colonizing CR-PA. 5
Materials and Methods
Preparation of the SuperCP medium
To design the composition of this medium (named SuperCP medium), the necessity to prevent contamination by gram-positive bacteria and fungi has been considered. The minimal inhibitory concentration (MIC) values of meropenem and imipenem defining resistance are, respectively, 16 and 8 mg/L for Pseudomonas spp. Based on our experience, 6 we have therefore tested several screening media and several concentrations of meropenem and imipenem for designing this novel screening medium. Optimal conditions were found by using the CHROMagar™ Pseudomonas medium (Chromagar, Paris, France) supplemented with 2 mg/L of meropenem (Table 1). For preparation of the SuperCP medium, the diluted powder of CHROMagar Pseudomonas was heated at 100°C for 15 min (and not at 125°C as for other nonchromogenic media). After cooling the medium for 1 hr at 56°C and addition of the antibiotic, plates were poured. The SuperCP plates were stored at 4°C and protected from direct light exposure for up to 1 week.
Composition of the SuperCP Screening Medium
With a volume of 1,000 mL of medium, 50 plates of 100 mm can be poured.
Tested strains
A total of 65 P. aeruginosa clinical isolates of worldwide origin were included in this study to evaluate the performance of the medium. In addition, a total of 22 so-called Pseudomonas-like strains were screened, including strains belonging to different species, namely Pseudomonas putida, Pseudomonas otitidis, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas alcaligenes, Pseudomonas oleovorans, Pseudomonas citronellolis, Pseudomonas cichorii, Pseudomonas viridiflava, Pseudomonas fragi, Pseudomonas balearica, Pseudomonas mosselii, and Pseudomonas tolaasii. Two other clinically relevant Pseudomonas-like species were included, namely Stenotrophomonas maltophilia and Burkholderia cepacia. The species were identified by API20E or Matrix Assisted Laser Desorption Ionization-Time of Flight. The carbapenemase content of all strains was characterized at the molecular level either by using PCR and sequencing, or whole-genome sequencing. In total, 29 meropenem-susceptible and 60 meropenem-nonsusceptible (meaning MIC values defined as susceptible, increase exposure (i), and resistant according to EUCAST version 10.0 guidelines) Pseudomonas spp. strains were used in our evaluation (Table 2). The MIC values of imipenem and meropenem were determined using E-test strips (bioMérieux, La Balme-les-Grottes, France) on Mueller-Hinton agar plates at 37°C according to EUCAST guidelines.
Minimal Inhibitory Concentrations of Carbapenems and Lowest Limits of Detection of SuperCP Screening Medium
Carbapenemases are boldened.
IMP, imipenem; MER, meropenem; NC, β-lactam resistance mechanism(s) not characterized, absence of carbapenemase.
Spiking experiments
Starting with an optical density of 0.5 McFarland standard (inoculum of ∼1.5 × 108 CFU/mL), serial 10-fold dilutions up to 1 × 102 CFU/mL were made in 0.85% saline solution. The strains were tested with spiked stools using this selective culture medium. Spiked fecal samples were made by adding 100 μL of serial-fold bacterial dilutions to 900 μL of stool suspension. Stool suspensions were obtained by suspending 6 g of freshly pooled feces from healthy volunteers in 50 mL of distilled water as described previously. 6 Aliquots of 100 μL of stool suspensions without addition of bacterial strains plated onto the SuperCP medium were used as negative controls.
Colony counting
For each 10-fold dilution, colonies were counted after overnight incubation. Experiments were performed at least in triplicates and results are presented in average. Sensitivity was calculated by the total of true positive over number of true positives + number of false negatives. Specificity is calculated by the total of true negative over true negatives + false positives.
Resistant strains used as positive controls were those showing MICs of meropenem greater or equal to 2 mg/L or MICs of imipenem greater than 4 mg/L, respectively. The sensitivity and specificity cutoff values for the detection of carbapenem-nonsusceptible Pseudomonas spp. were set at 1 × 103 CFU/mL, that is, the carbapenem-nonsusceptible Pseudomonas spp. isolates recovered on SuperCP medium plates at <1 × 103 CFU/mL were considered positive, while the meropenem-susceptible isolates grown when using an inoculum of ≥1 × 103 CFU/mL were considered negative as previously described. 7
Finally, to assess its storage stability, the meropenem-susceptible P. aeruginosa CIP100720 reference strain (MIC of meropenem at 0.38 μg/mL) was daily subcultured onto the SuperCP medium using a single batch of medium stored at 4°C.
Results
Most of the meropenem-nonsusceptible isolates could be recovered within 24 hr on the SuperCP medium plates using an inoculum below the cutoff value of 1 × 103 CFU/mL (being 1 × 100–1 × 102 CFU/mL). On the contrary, some meropenem-nonsusceptible isolates grew only when using an inoculum of >103 CFU/mL (the lowest limit of detection was above the cutoff value of 103).
The sensitivity and specificity of the SuperCP medium for screening carbapenem-nonsusceptible isolates were found to be at 91% and 100%, respectively, among Pseudomonas species.
No growth of the meropenem-susceptible P. aeruginosa CIP100720 reference strain was consistently observed during at least a 3-week period.
Discussion
The SuperCP medium constitutes an adequate screening medium to detect carbapenem-nonsusceptible Pseudomonas spp. isolates regardless of their resistance mechanism.
The sensitivity of detection was estimated at 92% with 5 out of 59 carbapenem-nonsusceptible strains growing at an higher inoculum than the detection limit cutoff (>103 CFU/mL). The MIC value interpretation of those strains was actually “susceptible increase exposure” (formerly intermediate) for meropenem, which agrees with the difficulty to detect them. All meropenem-resistant strains were correctly selected with this medium. On the contrary, the specificity was of 100%. Interestingly, this medium may also be used for screening non-P. aeruginosa nonfermenters such as S. maltophilia.
The possibility to have a selective medium for carbapenem-resistant Pseudomonas-like isolates is of clinical importance, since carbapenem resistance is of growing concern in those nonfermenter species. However, it is important to highlight that some carbapenemase-producing Enterobacterales strains could grow on this medium, with two out of six tested KPC-producing Klebsiella pneumoniae growing on the SuperCP medium (data not shown). Nevertheless, those K. pneumoniae strains could be differentiated from Pseudomonas spp. strains based on the colony color that is mauve or purple for K. pneumoniae instead of blue green for Pseudomonas spp.
Conclusion
The screening of potentially colonized patients was conducted to rapidly implement infection control measures, eventually limiting their spread. This medium is also adequate for epidemiological surveys aiming to evaluate the prevalence of those resistant bacteria from human and environmental specimen flora. Further clinical evaluation of this medium in daily clinical practice will be needed in particular for screening respiratory samples recovered from cystic fibrosis patients for whom nonfermenters such as S. maltophilia and P. aeruginosa are known to be important sources of pulmonary infections.
Footnotes
Authors' Contributions
We the authors declare that this article is original, has not been published before, and is not currently being considered for publication elsewhere. We confirm that the article has been read and approved by all the named authors and that there are no other persons who satisfied the criteria for authorship, who are not listed. We further confirm that the order of authors listed in the article has been approved by all of us. We understand that the Corresponding Author is the sole contact for the editorial process. He is responsible for communicating with the other authors about the progress, submissions of revisions, and final approval of proofs.
Disclosure Statement
No competing financial interests exist.
Funding Information
This work was funded by the Swiss National Science Foundation (projects number FNS-407240_177381 and FNS-407240_177382), and by the French Ministry of Agriculture (project IMMUNOCOLITEST).