10.17843/rpmesp.2020.374.5832
BRIEF REPORT

Extended-spectrum beta-lactamase-producing enterobacterales carrying the mcr-1 gene in Lima, Peru

Katherine Yauri-Condor ¹, Medical Technologist
Milagros Zavaleta Apestegui ¹, Molecular Biologist
Carlos Raúl Sevilla-Andrade ^1,2, Medical Technologist
Julia Piscoya Sara ², Medical doctor; ^d Master in Epidemiology
Claudia Villoslado Espinoza ³, Medical doctor; Specialist in Clinical Pathology
William Vicente Taboada ^3,4, Medical doctor; Specialist in Clinical Pathology
Edgar Gonzales-Escalante ^1,5, Medical Technologist; Master in Microbiology

¹ Centro de Investigaciones Tecnológicas, Biomédicas y Medioambientales - CITBM, Universidad Nacional Mayor de San Marcos, Lima, Perú.
² Instituto de Medicina Tropical «Daniel A. Carrión», Departamento Académico de Microbiología Médica, Facultad de Medicina, Universidad Nacional Mayor de San Marcos, Lima, Perú.
³ Instituto Nacional de Enfermedades Neoplásicas, Lima, Perú.
⁴ Laboratorios Unilabs, Lima, Perú.
⁵ Laboratorio de Resistencia Bacteriana, Instituto de Investigaciones en Bacteriología y Virología Molecular (IBaViM), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.

INTRODUCTION

The worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) gram-negative bacteria, including carbapenemase-producing enterobacterales (CPE), has led to the reinsertion of colistin as a last resort therapy; this antibiotic interacts directly with the outer lipopolysaccharide membrane ⁽¹⁾. The main resistance mechanisms involve the modification of lipid A, mediated by mutations in genes of the PhoPQ-PmrAB regulatory system, even during treatment of clinically relevant microorganisms such as Klebsiella pneumoniae ^(2,3). In 2015, a mechanism of plasmid-mediated colistin resistance (PMCR), related to the mcr-1 gene (Mobile Colistin Resistance) was described among enterobacterales isolated from animals and humans in China ⁽⁴⁾. This gene encodes a phosphoethanolamine transferase that modifies the colistin target, by adding phosphoethanolamine, which reduces the affinity for colistin ⁽⁵⁾.

Several studies have shown a worldwide distribution of mcr-1, especially in Escherichia coli, and occasionally in other bacterial species ⁽⁶⁾. As with other resistance genes, different allelic variants of mcr-1 have been detected (mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10) ^{(5, 6)}. The presence of mcr-1 genes has been reported from several countries in South America, in isolates obtained from humans, animals and food ^(7-9). In 2016, the World Health Organization (WHO) recommended implementing and strengthening the surveillance and epidemiological research of the PMCR ⁽¹⁰⁾. In Peru, the presence of mcr-1 in E. coli clinical isolates has been reported so far ^(11,12).

Therefore, this study is part of the project "Epidemiological surveillance of resistant bacteria in healthcare-associated infections" and is aimed at identifying the extended-spectrum beta-lactamase-producing enterobacterales (ESBL-PE) that carry the mcr-1 gene in Instituto Nacional de Enfermedades Neoplásicas (INEN) of Perú.

THE STUDY

This is a descriptive study, in which we collected 165 single consecutive isolates of ESBL-PE between January and December 2017. The samples included Escherichia coli (112), Klebsiella pneumoniae (41), Enterobacter cloacae (5), Proteus mirabilis (4), Klebsiella oxytoca (2) and Klebsiella ozaneae (1); recovered from blood samples (40), urine (57), lower respiratory secretions (12) and rectal swabs (56) from patients hospitalized at INEN.

Antimicrobial identification and measurement of susceptibility were carried out with the Phoenix M50 automated system. ESBLs were confirmed with the BD-Expert system (BD Diagnostics, Sparks, MD). We interpreted the results following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) ⁽¹³⁾. Colistin resistance was measured with Colistin Agar-Spot (CAS) screening (Mueller-Hinton Agar [Merck, Germany]), a method developed by the Antimicrobial Service, INEI ANLIS "Dr. Carlos G. Malbrán" ⁽¹⁴⁾ which uses colistin sulfate (Sigma-Aldrich, Germany). For detecting mcr gene, we used the phenotypic method of colistin pre-diffusion and inhibition with ethylenediaminetetraacetic acid (EDTA) (CPD-E) (Mueller-Hinton Agar [Merck, Germany]). This method was described by Yauri et al. ⁽¹⁵⁾ and uses 10 µg colistin discs (Oxoid, England); 372/900 µg and EDTA/SMA discs (Britannia, Argentina).

Total bacterial DNA was used as a mold for the molecular detection carried out in the laboratory of Molecular Epidemiology and Genetics of the Institute of Tropical Medicine "Daniel A. Carrión" in the Center for Technological, Biomedical and Environmental Research - CITBM of Universidad Nacional Mayor de San Marcos. For the identification of the resistance genes (mcr-1, bla_CTX-M, bla_NDM, and bla_KPC) we used the polymerase chain reaction (PCR) as described above ^(4,16,17) (Figure 1).

*CPD-E: pre-diffusion of colistin and inhibition with EDTA.

Figure 1. Detection of extended-spectrum beta-lactamase-producing enterobacterales carrying the mcr-1 gene.

This report has been conducted as part of the study by the Science and Technology Research Circles of the Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica (FONDECYT), approved by the INEN Research Department. This study follows the guidelines of good practice and ethics in biomedical research. The variables of interest were expressed as absolute and relative frequencies. We used Microsoft Excel for the descriptive analysis.

FINDINGS

Of the 165 ESBL-PE isolates, 25 (15.2%) were positive for the mcr-1 gene; 20 (12.1%) were resistant to colistin by the CAS screening method. Phenotypic detection of mcr with the CPD-E method had 100% correlation with the genotypic method (25 positive). The presence of the mcr-1 gene, per species, was as follows: E. coli (18), K. pneumoniae (4), E. cloacae (2) and K. oxytoca (1). Depending on the type of sample, mcr-1 was detected more frequently in isolates from rectal swabs (11/25) and in blood (9/25) (Table 1).

Table 1. Distribution of isolates of extended-spectrum beta-lactamase-producing enterobacterales carrying the mcr-1 gene by sample type and source.

In addition, the susceptibility profile of ESBL-PE carrying the mcr-1 gene showed resistance to fluoroquinolones and gentamicin and remained sensitive to amikacin. Notably, two isolates of K. pneumoniae showed resistance to carbapenems due to the presence of metalocarbapenemases (New Delhi Metal Beta-Lactamase [NDM]) (Figure 2).

AMK: amikacin; GEN: gentamicin; IPM: imipenem; MEM: meropenem; CAZ: ceftazidime; CTX: cefotaxime; CIP: ciprofloxacin.
* 22 isolates were carriers of bla_CTX-M genes, and 2 isolates were also carriers of bla_NDM genes.

Figure 2. Antimicrobial susceptibility profile of extended-spectrum beta-lactamase-producing enterobacterales carrying the mcr-1 gene (n = 25)*.

DISCUSSION

PMCR due to the mcr-1 gene has been widely reported throughout the world and in some cases has been related to other resistance markers, such as beta-lactamases (ESBL or carbapenemases) ^{(2,7,11,18-20)}. In our study, 15.2% of the isolates recovered from samples of different infections and colonizations (carriers) in hospitalized patients presented ESBL-PE carrying the mcr-1 gene. In 2017, Ugarte et al. ⁽¹²⁾ made the first Peruvian report about mcr-1 on seven E. coli isolates recovered from urine cultures of community patients. In 2019, Deshpande et al. ⁽¹¹⁾ reported three E. coli isolates producing mcr-1 recovered in Peru, 2016 from blood and skin infection samples, one of the isolates was a producer of ESBL_{(blaCTX-M-55)}.

The presence of beta-lactamase-producing enterobacterales carrying the mcr-1 gene is increasing in the region ^(18-20), not only co-producers of ESBL, but also of carbapenemases, as observed in two of our MDR K. pneumoniae isolates, with resistance ranging from beta-lactams (CTX-M, NDM), fluoroquinolones, gentamicin and even colistin, which is considered an antibiotic of last resort against MDR bacteria, leaving only amikacin as a therapeutic alternative.

The CAS screening method shows a 99.5% accuracy compared to the broth microdilution method, considered as the reference method for the determination of colistin resistance. In our study, colistin resistance was detected in 17/25 mcr-1-producing isolates, probably because these isolates had a minimum inhibitory concentration (MIC) <3 µg/mL, which is the colistin concentration used by the CAS screening method ⁽¹⁴⁾.

A K. pneumoniae isolate classified as colistin resistant did not carry the mcr-1 gene; its resistance to colistin could be related to mutations in genes of the PhoPQ-PmrAB regulatory system ^(2,3); or to the presence of allelic variants of the mcr gene not investigated in this study ^(5,6). In 2020, the CLSI changed the cut-off points for the interpretation of colistin in enterobacterales, Pseudomonas aeruginosa and Acinetobacter spp., which can now only be classified in two categories: intermediate (≤2 µg/mL) and resistant (≥4 µg/mL). The CLSI also specifies that the only acceptable methodologies are broth microdilution for colistin (BMD), colistin agar test (CAT) and colistin broth disc elution (CBDE) ⁽¹³⁾.

The CPD-E method ⁽¹⁵⁾ is based on the property of the zinc-dependent metalloprotein of the phosphoethanolamine transferase encoded by the mcr-1 gene, which allows it to be inhibited by chelators, such as EDTA. Therefore CPD-E method had an optimal correlation with mcr-1 gene carrier isolates, proving to be a good phenotypical alternative for detecting MCR producers.

The presence of the mcr-1 gene in clinical MDR isolates that caused infections is alarming, the gastrointestinal presence of bacteria with this gene has already been demonstrated in hospital or community environment ⁽¹⁰⁾. In our study, the frequency of colonization with ESBL‑PE carriers of mcr-1 gene was 44%; this colonization is very dangerous, since plasmids may be the cause of its dissemination to other to other virulent strains or epidemic clones ⁽¹⁰⁾.

This study has some limitations. The results obtained correspond to a collection of MDR isolates (producers of ESBL) from a specialized health institution and cannot be extrapolated to other institutions. It is necessary to evaluate the presence of mcr-1 gene with a higher number of non-MDR isolates from different hospitals to know the real impact of this resistance marker in our country. Besides, we did not analyze allelic variants different from the mcr-1 gene and other causes of resistance to colistin, such as mutations in the genes of the regulatory system of lipid A from the bacterial membrane.

In conclusion, data collection on bacterial resistance to last-resort antimicrobials is crucial to establish policies according to the local context and to compare them at regional and global level. Although our results show the panorama of a single health institution, the appearance of MDR isolates with colistin resistance supports the need to carry out molecular epidemiology studies to prevent the establishment of healthcare-associated infections by this type of microorganisms.

Acknowledgements: To the staff of the Laboratory of Microbiology of Instituto Nacional de Enfermedades Neoplásicas for their collaboration in the process of collecting the isolates; to the research team of Biotechnology and Health of Centro de Investigaciones Tecnológicas, Biomédicas y Medioambientales for their contribution in the molecular biology; and to the staff of the Laboratory of Molecular Epidemiology and Genetics of Universidad Nacional Mayor de San Marcos for their continuous support.

Authorship contributions: KYC and EGE collected data and samples, and conceived the article. MZA, CRSA and JPS contributed to the idea for the research, as well as to the writing, and technical and administrative assistance. CBE and WVT participated in the data and study material collection and writing of the article. All authors conducted the critical review of the article, approved the final version and assumed responsibility for the contents of the manuscript.

Conflicts of Interest: The authors have no conflict of interest to declare.

Funding: Study funded by FONDECYT for the Science and Technology Research Circles (RD No. 142-2015-FONDECYT-DE) and by the Centro de Investigaciones Tecnológicas, Biomédicas y Medioambientales - CITBM.

REFERENCES

1. Falagas ME, Kasiakou SK. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis. 2005;40(9):1333-41. doi: 10.1086/429323.

2. Gao R, Hu Y, Li Z, Sun J, Wang Q, Lin J, et al. Dissemination and Mechanism for the MCR-1 Colistin Resistance. PLoS Pathog. 2016;12(11):e1005957. doi: 10.1371/journal.ppat.1005957.

3. Blair JM, Webber MA, Baylay AJ, Aqbolu DO, Piddock LJ. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol. 2015;13(1):42-51. doi: 10.1038/nrmicro3380.

4. 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;16(2):161–168. doi: 10.1016/S1473-3099(15)00424-7.

5. Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev. 2017;30(2):557-96. doi: 10.1128/CMR.00064-16.

6. Giamarellou H. Epidemiology of infections caused by polymyxin-resistant pathogens. Int J Antimicrob Agents. 2016;48(6):614-21. doi: 10.1016/j.ijantimicag.2016.09.025.

7. Quiroga C, Nastro M, Di Conza J. Current scenario of plasmid-mediated colistin resistance in Latin America. Rev Argent Microbiol. 2018;51(1):93-100. doi: 10.1016/j.ram.2018.05.001.

8. Fernandes MR, Moura Q, Sartori L, Silva KC, Cunha MP, Esposito F, et al. Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene. Euro Surveill. 2016;21(17):30214. doi: 10.2807/1560-7917.ES.2016.21.17.30214.

9. Monte DF, Mem A, Fernandes MR, Cerdeira L, Esposito F, Galvao JA, et al. Chicken meat as a reservoir of colistin resistant Escherichia coli strains carrying mcr-1 genes in South America. Antimicrob Agents Chemother. 2017;61(5):e02718-16. doi: 10.1128/AAC.02718-16.

10. Organización Panamericana de la Salud/Organización Mundial de La Salud [internet]. Alerta Epidemiológica: Enterobacterias con resistencia transferible a colistín, implicaciones para la salud pública en las Américas [Internet]. Washington D.C.: OPS/OMS; 2016. [citado el 25 de abril de 2020]. Disponible en: https://www.paho.org/hq/dmdocuments/2016/2016-jun-10-alerta-epi-enterob-resist.pdf.

11. Deshpande LM, Hubler C, Davis AP, Castanheira M. Updated prevalence of mcr-like genes among Escherichia coli and Klebsiella pneumoniae in the SENTRY program and characterization of mcr-1.11 variant. Antimicrob Agents Chemother. 2019;63:e02450-18. doi: 10.1128/AAC.02450-18.

12. Ugarte R, Olivo J, Corso A, Pasteran F, Albornoz E, Sahuanay ZP. Resistencia a colistín mediado por el gen mcr-1 identificado en cepas de Escherichia coli y Klebsiella pneumoniae. Primeros reportes en el Perú. An Fac med. 2018;79(3):213-7. doi: 10.15381/anales. v79i3.15313.

13. Clinical and Laboratory Standards Institute (CLSI). 2020. Performance standards for antimicrobial susceptibility testing: 30th informational supplement M100-S30 [Internet]. CLSI, Wayne, PA [citado el 25 de abril de 2020]. Disponible en: https://clsi.org/standards/products/microbiology/documents/m100/.

14. Servicio Antimicrobianos, Laboratorio Nacional de Referencia en Antimicrobianos, INEI-ANLIS "Dr Carlos G. Malbrán" [Internet]. Métodos de Screening "colistín Agar-Spot". [citado el 25 de abril de 2020]. Disponible en: http://antimicrobianos.com.ar/ATB/wp-content/uploads/2017/09/Protocolo-Agar-spot-COL-2017-version2-Agosto2017.pdf.

15. Yauri K, Gonzales E, Di Conza J, Gutkind G. Detection of plasmid-mediated colistin resistance by colistin pre-diffusion and inhibition with EDTA test (CPD-E) in Enterobactereaceae. J Microbiol Methods. 2019;167:105759. doi: 10.1016/j.mimet.2019.105759.

16. Servicio Antimicrobianos, Laboratorio Nacional de Referencia en Antimicrobianos, INEI-ANLIS "Dr Carlos G. Malbrán" [Internet]. Protocolo de PCR-Multiplex para la detección de carbapenemasas. [citado el 25 de abril de 2020]. Disponible en: http://antimicrobianos.com.ar/2019/10/protocolo-de-pcr-multiplex-para-la-deteccion-de-carbapenemasas/.

17. Ghasemi Y, Archin T, Kargar M, Mohkam M. A simple multiplex PCR for assessing prevalence of extended-spectrum β-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz, Iran. Asian Pac J Trop Med. 2013;6(9):703-708. doi: 10.1016/S1995-7645(13)60122-4.

18. Sellera FP, Fernandes MR, Sartori L, Carvalho MPN, Esposito F, Nascimento CL, et al. Escherichia coli carrying IncX4 plasmid mediated mcr-1 and blaCTX-M genes in infected migratory Magellanic penguins (Spheniscus magellanicus). J Antimicrob Chemother. 2017;72(4):1255-6. doi: 10.1093/jac/dkw543.

19. Delgado-Blas JF, Ovejero CM, Abadia-Patino L, Gonzalez-Zorn B. Coexistence of mcr-1 and blaNDM-1 in Escherichia coli from Venezuela. Antimicrob Agents Chemother. 2016;60(10):6356-8. doi: 10.1128/AAC.01319-16.

20. Dalmolin TV, Martins AF, Zavascki AP, de Lima-Morales D, Barth AL. Acquisition of the mcr-1 gene by a high-risk clone of KPC-2-producing Klebsiella pneumoniae ST437/CC258, Brazil. Diagn Microbiol Infect Dis. 2018;90(2):132-3. doi: 10.1016/j.diagmicrobio.2017.09.016.

Correspondence: Edgar Gonzales Escalante; Calle José Santos Chocano 199, Ciudad Universitaria, Bellavista, Callao; egones_5@hotmail. com

Cite as: Yauri-Condor K, Zavaleta Apestegui M, Sevilla-Andrade CR, Piscoya Sara J, Villoslado Espinoza C, Vicente Taboada W, et al. Extended-spectrum beta-lactamase-producing enterobacterales carrying the mcr-1 gene in Lima, Peru. Rev Peru Med Exp Salud Publica. 2020;37(4). doi: https://doi.org/10.17843/rpmesp.2020.374.5832.