Carriage of two carbapenem-resistance genes in Pseudomonas aeruginosa isolated from hospital-acquired infections in children from Costa Rica: the importance of local epidemiology

Background The assessment of Hospital-acquired infections due to multidrug-resistant bacteria involves the use of a variety of commercial and laboratory-developed tests to detect antimicrobial resistance genes in bacterial pathogens; however, few are evaluated for use in low- and middle-income countries. Methods We used whole-genome sequencing, rapid commercial molecular tests, laboratory-developed tests and routine culture testing. Results We identified the carriage of the metallo-β-lactamase blaVIM-2 and blaIMP-18 alleles in Carbapenem-Resistant Pseudomonas aeruginosa infections among children in Costa Rica. Conclusions The blaIMP-18 allele is not present in the most frequently used commercial tests; thus, it is possible that the circulation of this resistance gene may be underdiagnosed in Costa Rica.


Background
The global increase of infections due to multidrug-resistant bacteria remains a public health and sustainable development problem [1]. The global antimicrobial resistance surveillance system (GLASS) encourages healthcare authorities to increase laboratory capacity. To improve surveillance and diagnostic stewardship, GLASS also recommends the implementation of rapid, accurate diagnostic testing for antibiotic resistance [2]. The methods for the detection of resistance mechanisms are diverse, ranging from phenotypic to more complex genotypic tests, and whole-genome sequencing (WGS). The continuous development of diagnostic tests in and for high-income countries makes them available for other countries at a good price. However, information on antimicrobial resistance in low-and middle-income countries is scarce, as is the performance of these kits in different epidemiological contexts. Carbapenem-Resistant Pseudomonas aeruginosa (CRPA) is frequently isolated from healthcare-associated infections (HAI). The mechanisms of CRPA resistance can be driven by porin loss, efflux pump activity, or by horizontal gene transfer encoded in plasmids [3]. In Costa Rica, the National Children's Hospital is a tertiary referral hospital within the socialized medical care system. HAI assessment has Open Access *Correspondence: cperezc@ccss.sa.cr División de Microbiología, Laboratorio Clínico, Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera", Centro de Ciencias Médicas de La Caja Costarricense del Seguro Social, PO Box: 1654-1000, San José, Costa Rica recently improved in this hospital with the implementation of a diagnostic stewardship program, which was established according to its needs and resources. To better understand CRPA circulation in Costa Rica and compare detection methods, we studied P. aeruginosa isolated from pediatric patients with HAI between late 2018 and 2020 using conventional phenotypic methods, rapid molecular test, and WGS.

Results
A total of 8 out of the 32 bacterial isolates identified as P. aeruginosa exhibited resistance to imipenem and meropenem by automated means. In all 8 strains, mCIM was positive, indicating carbapenemase activity. Furthermore, MBL E-test methods confirmed the carbapenem resistance due to the presence of metallo-β-lactamase in all strains. The molecular methods classified all 8 strains in agreement with the observed phenotypic assays. However, GenXpert Carba RT-PCR, as well as the reversehybridization assay showed positive results for bla VIM detection only. Conventional PCR using LDT detected both bla VIM and bla IMP genes. The analyses of resistance genes using the WGS data (ABRicate) [11] identified alleles bla VIM-2 and bla IMP-18 . Taken together, 4 out of the 8 strains exhibited both bla VIM-2 and bla IMP-18 alleles. The remaining 4 strains harbored only the bla VIM-2 gene.

Discussion
Carbapenems constitute one of the final lines of defense against resistant bacteria, particularly Gram-negative bacilli. CRPA is ranked as "critical priority" for research and development for new drugs and to implement antimicrobial stewardship initiatives by the World Health Organization [13]. Detecting the mechanisms of resistance to antimicrobials provide a helpful tool to prevent and control HAIs, especially considering the growing evidence of mobile genetic elements mediating and exacerbating nosocomial outbreaks [14,15]. To address these critical pathogens, we implemented commercial rapid molecular tests, hybridization assays, and whole-genome sequencing to analyze CRPA in HAI and compare the aforementioned methods.
Eight of the 32 P. aeruginosa isolates from HAI in the National Children's Hospital of Costa Rica exhibited carbapenem resistance. The presence of the bla VIM gene was identified using two different commercial kits. However, when testing the same isolates by PCR LDT, we also detected the bla IMP gene in addition to the bla VIM gene. WGS analyses confirmed the presence of alleles bla VIM-2 and bla IMP-18 , which were previously described in one of the main hospitals treating adults in the country [16]. Both genes are known to encode metallo-β-lactamase that confer resistance to carbapenems. Our analyses provide valuable information about the circulation of P. aeruginosa carrying bla IMP-18 and bla VIM-2 alleles in pediatric infections in Costa Rica. Moreover, we identified that allele bla IMP-18 is present in HAI, but not targeted in the current rapid molecular technologies available in Costa Rica. This could lead to miss-identification of the P. aeruginosa resistance mechanisms if healthcare facilities don't have access to redundant methods for confirmation.
This work brings together the context of hospitalacquired infections, antimicrobial stewardship, diagnostic stewardship, availability of diagnostic methods, and whole genome sequencing to highlight the importance of local epidemiology when adopting strategies to fight antimicrobial resistance. Similar situations can be observed globally as infections due to multidrug-resistant bacteria increase and new technologies become available. Rapid molecular tests for the detection of antibacterial resistance continue to expand around the world. The availability, presentation, multiplex format, international validation, and low price makes these kits very attractive to use in economically constrained countries. However, they are developed for high-income countries in accordance with their needs and epidemiological contexts; thus, they might not have all of the targets that are circulating in other countries.

Conclusions
The results of this study highlight the importance of knowing the local epidemiology when monitoring for CRPA in the context of HAIs using rapid molecular tests that were originally created for a different country.