To the Editor. Since its emergence, isolates of carbapenem-resistant Klebsiella pneumoniae have been disseminated in hospitals in different regions of Peru. Currently, carbapenemase type New Delhi metallo-beta-lactamase (NDM) is the most frequently detected among K. pneumoniae and Escherichia coli isolates ¹1. Mayta-Barrios M, Ramirez-Illescas J, Pampa-Espinoza L, Yagui-Moscoso M. Caracterización molecular de carbapenemasas en el Perú durante el 2019. Rev Per Med Exp Salud Publica. 2021;38(1):113-8. doi: 10.17843/rpmesp.2021.381.5882.
https://doi.org/10.17843/rpmesp.2021.381... . In these infections, colistin is often the antibiotic of last resort, since new antibiotics against these pathogens are not included in Peru’s National Drug Formulary.

In order to assess colistin susceptibility, the recommended method for estimating the minimum inhibitory concentration is broth microdilution, but this method is not practical for most microbiology laboratories. In contrast, the disk elution method uses reagents and equipment usually available in routine laboratory processes and is less time consuming. This method is also recommended by the Clinical and Laboratory Standards Institute (CLSI) for the evaluation of colistin in Enterobacteriaceae²2. Clinical and Laboratory Standards Institute. (2018). Performance standards for antimicrobial susceptibility testing; 33rd ed. CLSI standard M100. Wayne, PA: CLSI; 2021..

The mechanisms of colistin resistance among gram-negative bacilli include mainly resistance genes located in chromosomes or in transferable elements (plasmids or transposons), the latter being the most widespread mechanism. The mcr-1 gene, including its variants and sub-variants that are carried on these plasmids or transposons have been detected worldwide among several species of gram-negative bacteria either in humans, animals or in the environment. The mcr genes encode a transferase that increases the cationic charge of the lipopolysaccharide causing decreased binding of colistin to the lipopolysaccharide ³3. El-Sayed Ahmed MAEG, Zhong LL, Shen C, Yang Y, Doi Y, Tian GB. Colistin and its role in the era of antibiotic resistance: an extended review (2000-2019). Emerg Microbes Infect. 2020;9(1):868-85. doi: 10.1080/22221751.2020.1754133.
https://doi.org/10.1080/22221751.2020.17... .

We evaluated colistin susceptibility in 317 isolates of E. coli (n=199) and K. pneumoniae (n=118) obtained from blood cultures. These samples were collected during an antimicrobial resistance surveillance study that included 15 hospitals in 12 regions of Peru between 2017-2019 ⁴4. Krapp F, García C, Hinostroza N, Astocondor L, Rondon CR, Ingelbeen B, et al. Prevalence of antimicrobial resistance in Gram-Negative bacteria bloodstream infections in Peru and associated outcomes: VIRAPERU Study. Am J Trop Med Hyg. 2023; Sep 18:tpmd220556. doi: 10.4269/ajtmh.22-0556.
https://doi.org/10.4269/ajtmh.22-0556... . Bacterial identification was performed through conventional biochemical tests and susceptibility to carbapenems tests were performed through the disc-diffusion method. In order to assess susceptibility to colistin, the disk elution technique was performed following the procedures described by CLSI ²2. Clinical and Laboratory Standards Institute. (2018). Performance standards for antimicrobial susceptibility testing; 33rd ed. CLSI standard M100. Wayne, PA: CLSI; 2021., identifying 9.1% (29/317) isolates resistant to colistin (≥4 μg/mL), of which 7.5% (15/199) and 11.9% (14/118) were E. coli and K. pneumoniae isolates, respectively (Table 1). Regarding resistance to carbapenems, 11.0% of K. pneumoniae isolates were resistant to some carbapenemics, on the other hand, none of the E. coli isolates were found to be resistant.

We evaluated the presence of the mcr-1 gene by conventional PCR technique in all colistin-resistant isolates. The primers CLR-5F and CLR-5R described by Liu et al. ⁵5. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;6(2):161-8. doi: 10.1016/S1473-3099(15)00424-7.
https://doi.org/10.1016/S1473-3099(15)00... ⁾ were used. The master mix was prepared to a final volume of 25 μl with 0.2 μM of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, 1X buffer, 0.03 U/μl of GOTAQ® G2 FLEXI DNA polymerase (Promega, Madison, USA) and 2 μl of DNA. The cycling conditions described by Faccone et al. were used with a modification in the hybridization temperature of 72 °C for 5 min. The size of the amplicons was 309 base pairs.

The mcr-1 gene was detected in 9 of the 29 colistin-resistant isolates, 8/15 in E. coli isolates and 1/14 in K. pneumoniae isolates (Table 1). These nine isolates were susceptible to carbapenems and were distributed in 7 of the 15 hospitals in 5 of the 12 evaluated regions (Lima, La Libertad, Ancash, Loreto and Puerto Maldonado).

The epidemiology of Enterobacteriaceae species carrying the mcr-1 gene in Peru and other Latin American countries may be underestimated since colistin resistance testing is not routinely performed on all gram-negative bacteria, and in most cases, it is reserved for those bacteria that are resistant to carbapenems. Yauri et al. showed that 15.2% of extended-spectrum beta-lactamase-producing enterobacteria were positive for detection of the mcr-1 gene in a hospital in Lima ⁶6. Yauri-Condor K, Apestegui MZ, Sevilla-Andrade CR, Sara JP, Espinoza CV, Taboada WV, et al. Extended-spectrum beta-lactamase producing enterobacterales carrying the mcr-1 gene in Lima, Peru. Rev Per Med Exp Salud Publica. 2020;37(4):711-5. doi: 10.17843/rpmesp.2020.374.5832.
https://doi.org/10.17843/rpmesp.2020.374... . In our study, all isolates carrying the mcr-1 gene were susceptible to carbapenems.

In conclusion, the presence of Enterobacteriaceae species carrying the mcr-1 gene causing bloodstream infections distributed in several hospitals and regions of Peru, alerts about the dissemination of resistance to this last-resort antibiotic in our country. Other chromosome-mediated mechanisms of colistin resistance should be explored since we found that the mcr-1 gene was present in less than one third of colistin-resistant bacteria.

Acknowledgments.

To all the collaborators of the 15 hospitals that participated in the study.

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