ABSTRACT

Objective.

Colistin is an antibiotic of last resort for treating serious Gram-negative bacterial infections. However, the misuse of colistin, especially as an animal growth promoter, has contributed to increasing antimicrobial resistance, mediated mainly through plasmid transfer of the mcr-1 gene. This study assessed the prevalence of phenotypic and molecular colistin resistance in Escherichia coli and Klebsiella pneumoniae in Ecuador in healthy humans and their chickens and pigs.

Methods.

Fecal samples were collected from humans and their chickens and pigs in two rural coastal and Amazon regions between April and August 2020. Gram-negative bacteria were isolated and identified using conventional techniques. Phenotypic resistance was determined using the broth microdilution technique, and the mcr-1 gene was detected using conventional polymerase chain reaction.

Results.

A total of 438 fecal samples were obtained from 137 humans, 147 pigs and 154 chickens. The prevalence of E. coli isolates was 86.3% (378/438) and K. pneumoniae, 37.4% (164/438). Overall, the mcr-1 gene was found in 90% (340/378) of E. coli isolates, with higher prevalences found in isolates from coastal regions (96.5%, 191/198), humans (95.6%, 111/116) and chickens (91.8%, 123/134); for K. pneumoniae, the gene was found in 19.5% (32/164) of isolates, with equal distribution between regions and hosts. Only four isolates, two E. coli and two K. pneumoniae, showed phenotypic resistance: mcr-1 was present in both E. coli strains but absent in the K. pneumoniae strains.

Conclusions.

Despite a low prevalence of phenotypic resistance to colistin, the high prevalence of the mcr-1 gene in E. coli is of concern. Ecuador’s ban on using colistin in animal husbandry must be enforced, and continual monitoring of the situation should be implemented.

Keywords
Colistin; Escherichia coli; Klebsiella pneumoniae; humans; animals; drug resistance; genes, MDR; operational research; Ecuador

RESUMEN

Objetivo.

La colistina es un antibiótico de último recurso para tratar infecciones graves por bacterias gramnegativas. Sin embargo, su uso indebido, especialmente para estimular el crecimiento animal, ha contribuido con el aumento de la resistencia a los antimicrobianos, mediada principalmente por la transferencia de plásmidos del gen mcr-1. En este estudio se evaluó la prevalencia de la resistencia fenotípica y molecular a la colistina de las bacterias Escherichia coli y Klebsiella pneumoniae en humanos sanos, sus pollos y cerdos en Ecuador.

Métodos.

Se recolectaron muestras fecales de humanos, así como de sus pollos y cerdos, en dos zonas rurales de la región costera y la región amazónica entre abril y agosto del 2020. Se aislaron las bacterias gramnegativas y se identificaron empleando técnicas convencionales. Se determinó la resistencia fenotípica mediante la técnica de microdilución en caldo y se detectó el gen mcr-1 con la técnica convencional de reacción en cadena de la polimerasa.

Resultados.

Se obtuvo un total de 438 muestras fecales de 137 humanos, 147 cerdos y 154 pollos. La prevalencia de E. coli en las cepas aisladas fue del 86,3% (378/438) y la de K. pneumoniae, del 37,4% (164/438). En general, se detectó el gen mcr-1 en el 90% (340/378) de las cepas aisladas de E. coli y la mayor prevalencia encontrada fue en cepas aisladas de la región costera (96,5%, 191/198), humanos (95,6%, 111/116) y pollos (91,8%, 123/134); en el caso de K. pneumoniae, el gen se encontró en el 19,5% (32/164) de las cepas, con una distribución equitativa entre regiones y hospedadores. Únicamente cuatro cepas aisladas, dos de E. coli y dos de K. pneumoniae, mostraron resistencia fenotípica: el gen mcr-1 estaba presente en ambas cepas de E. coli y ausente en las cepas de K. pneumoniae.

Conclusiones.

Si bien hubo una baja prevalencia de resistencia fenotípica a la colistina, la alta prevalencia del gen mcr-1 en E. coli es preocupante. Es necesario hacer cumplir la prohibición del uso de colistina en la cría de animales en Ecuador, así como realizar un seguimiento continuo de la situación.

Palabras clave
Colistina; Escherichia coli; Klebsiella pneumoniae; humanos; animales; resistencia a medicamentos; genes MDR; investigación operativa; Ecuador

RESUMO

Objetivo.

A colistina é um antibiótico de último recurso para o tratamento de infecções graves por bactérias Gram-negativas. Entretanto, o uso indevido da colistina, principalmente como promotor de crescimento animal, tem contribuído para o aumento da resistência a antimicrobianos, principalmente por transferência horizontal do gene mcr-1 mediada por plasmídeos. Este estudo avaliou a prevalência de resistência fenotípica e molecular à colistina em Escherichia coli e Klebsiella pneumoniae no Equador em humanos hígidos e em galinhas e porcos por eles criados.

Métodos.

Entre abril e agosto de 2020, foram coletadas amostras de fezes de habitantes de duas regiões litorâneas e amazônicas do Equador e de galinhas e porcos por eles criados. Bactérias Gram-negativas foram isoladas e identificadas por meio de técnicas convencionais. A resistência fenotípica foi determinada pela técnica de microdiluição em caldo, e o gene mcr-1 foi detectado por reação em cadeia da polimerase convencional.

Resultados.

Foram obtidas 438 amostras fecais de 137 humanos, 147 suínos e 154 galinhas. A prevalência de isolados de E. coli foi de 86,3% (378/438), e de K. pneumoniae, 37,4% (164/438). Em geral, o gene mcr-1 foi encontrado em 90% (340/378) dos isolados de E. coli, com maiores prevalências encontradas em isolados de regiões litorâneas (96,5%, 191/198), humanos (95,6%, 111/116) e galinhas (91,8%, 123/134); para K. pneumoniae, o gene foi encontrado em 19,5% (32/164) dos isolados, com igual distribuição entre regiões e hospedeiros. Somente quatro isolados, dois de E. coli e dois de K. pneumoniae, demonstraram resistência fenotípica: o gene mcr-1 estava presente em ambas as cepas de E. coli, mas ausente nas de K. pneumoniae.

Conclusões.

Apesar da baixa prevalência de resistência fenotípica à colistina, a alta prevalência do gene mcr-1 em E. coli é preocupante. É preciso fiscalizar a proibição ao uso agropecuário de colistina no Equador e implementar o monitoramento contínuo da situação.

Palavras-chave
Colistina; Escherichia coli; Klebsiella pneumoniae; humanos; animais; resistência a medicamentos; genes MDR; Equador

Antimicrobial resistance is recognized as one of the most serious global threats to human health. The emergence of multidrug resistance – defined as an organism showing resistance to three or more classes of antibiotics – in Escherichia coli and Klebsiella pneumoniae is particularly concerning (¹1. Hee Park S. Management of multi-drug resistant organisms in healthcare settings. J Korean Med Assoc. 2018;61(1):26–35.). Both are Gram-negative bacteria that cause serious infections and have multiple resistance mechanisms. The commonest examples of these are extended-spectrum β-lactamases, carbapenemases (²2. Bastidas-Caldes C, Romero-Alvarez D, Valdez-Vélez V, Morales RD, Montalvo-Hernández A, Gomes-Dias C, et al. Extended-spectrum beta-lactamases producing Escherichia coli in South America: a systematic review with a One Health perspective. Infect Drug Resist. 2022;15:5759–79.) and for colistin, the presence of the mcr-1 gene.

A recent study has highlighted the growing threat of resistance to colistin mediated through mcr genes, with documented examples of resistance in animals, humans, food and the environment (³3. Elbediwi M, Li Y, Paudyal N, Pan H, Li X, Xie S, et al. Global burden of colistin-resistant bacteria: mobilized colistin resistance genes study (1980–2018). Microorganisms. 2019;7(10):1–18.). Colistin is an antibiotic considered one of the medicines of last resort against multidrug-resistant Gram-negative bacterial infections (⁴4. WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance. Critically important antimicrobials for human medicine: Ranking of medically important antimicrobials for risk management of antimicrobial resistance due to non-human use, sixth revision. Geneva: World Health Organization; 2019. https://www.who.int/publications/i/item/9789241515528
https://www.who.int/publications/i/item/... ). Colistin is commonly used in animal husbandry for treatment and as a growth promoter in food supplements, either in normal or excessively high doses, thus increasing the selection for colistin resistance in animals (⁵5. 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.). In addition, the effectiveness of antibiotics, including colistin, is being threatened by their excessive use in veterinary medicine. Indeed, in some countries, the use of colistin has been 600 times higher in animals than in humans (⁶6. Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill. 2016;21(9):30155.).

In 2016, Liu et al. identified in China for the first time a plasmid-mediated colistin- resistance gene, a mobile element that contains the mcr-1 gene (⁷7. 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):161–8.). This gene encodes the expression of phosphoethanolamine transferase, affecting lipid A, which confers antibiotic resistance in Enterobacterales (⁷7. 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):161–8.). Colistin-resistant Enterobacterales, particularly E. coli and K. pneumoniae, carrying the mcr-1 gene have since been reported worldwide, including in the Americas (in Argentina, Bolivia [Plurinational State of], Brazil, Colombia, Peru and the United States) (⁸8. Bastidas-Caldes C, de Waard JH, Salgado MS, Villacís MJ, Coral-Almeida M, Yamamoto Y, et al. Worldwide prevalence of mcr-mediated colistin-resistance Escherichia coli in isolates of clinical samples, healthy humans, and livestock—a systematic review and meta-analysis. Pathogens. 2022;11(6):659.). Studies in Latin America have suggested that this spread might be associated with the horizontal transfer of colistin-resistance genes (⁹9. Hoa HTT, Higashi A, Yamaguchi T, Kawahara R, Calvopina M, Bastidas-Caldés A, et al. Fusion plasmid carrying the colistin resistance gene mcr of Escherichia coli isolated from healthy residents. J Glob Antimicrob Resis. 2022;30:152–4.). Since the discovery of the mcr-1 gene and its presence in Enterobacterales plasmids, worldwide surveillance of colistin resistance has been strengthened (⁵5. 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.).

In 2016, Ortega-Paredes et al. reported the first clinical isolate of colistin-resistant E.coli harboring the mcr-1 gene in Ecuador in an adolescent with appendicitis (¹⁰10. Ortega-Paredes D, Barba P, Zurita J. Colistin-resistant Escherichia coli clinical isolate harbouring the mcr-1 gene in Ecuador. Epidemiol Infect. 2016;144(14):2967–70.). Since then, studies of animals on rural farms where there is extensive use of colistin as a growth promoter have shown widespread distribution of colistin-resistant E.coli, with a high proportion of isolates containing mcr-1 genes (¹¹11. Albán MV, Núñez EJ, Zurita J, Villacís JE, Tamayo R, Sevillano G, et al. Canines with different pathologies as carriers of diverse lineages of Escherichia coli harbouring mcr-1 and clinically relevant β-lactamases in central Ecuador. J Glob Antimicrob Resist. 2020;22:182–3.–¹³13. Montero L, Irazabal J, Cardenas P, Graham JP, Trueba G. Extended-spectrum beta-lactamase producing-Escherichia coli isolated from irrigation waters and produce in Ecuador. Front Microbiol. 2021;12:709418.). With the growing importance of the One Health concept, documenting the prevalence of colistin-resistance genes in both humans and animals in Ecuador is critical to establish a baseline of how far the mcr genes have dispersed (¹⁴14. Bastidas-Caldes C, Ochoa J, Guerrero-Latorre L, Moyota-Tello C, Tapia W, Rey-Pérez JM, et al. Removal of extended-spectrum beta-lactamase-producing Escherichia coli, ST98, in water for human consumption by black ceramic water filters in low-income Ecuadorian highlands. Int J Environ Res Public Health. 2022;19(8):4736.). This information will help to reinforce national surveillance and guide policies for the rational use of colistin. Such a study will also provide evidence about the possible cross-transfer of mcr-1 genes between different host species. Therefore, this study aimed to assess the prevalence of phenotypic colistin resistance and the presence of mcr-1 in E. coli and K. pneumoniae from humans and their backyard animals (chickens and pigs) living in selected rural communities in Ecuador between April and August 2020.

MATERIALS AND METHODS

Study design and sampling

This was a cross-sectional study using primary data and conducted in rural areas of Ecuador.

Ecuador is a country that straddles the equator on South America’s west coast. It has a diverse landscape that includes the Amazon jungle, Andean highlands, a coastal region and the wildlife-rich Galapagos Islands. Rural hamlets in the coastal region (province of Santo Domingo de los Tsáchilas) and the Amazon region (province of Pastaza) were included in the study. Each hamlet had a population of around 700 inhabitants in about 140 households, with a mean household size of 5 persons. There were approximately 15 hamlets and farmhouses in each of the study regions. Most inhabitants earn their livelihood from agriculture and farming activities, with every family usually having chickens, pigs, ducks and some cattle in their backyard. These hamlets were selected because there was a preliminary report disclosing information about resistance to colistin and cefotaxime in E. coli in backyard animals in these regions (¹²12. Yamamoto Y, Calvopina M, Izurieta R, Villacres I, Kawahara R, Sasaki M, et al. Colistin-resistant Escherichia coli with mcr genes in the livestock of rural small-scale farms in Ecuador. BMC Res Notes. 2019;12(1):121.).

Human fecal samples were collected and placed in semisolid Cary–Blair transport medium for coliforms (Eiken Chemical, Tokyo, Japan). For samples from backyard animals, cloacal and rectal swabs were taken from, respectively, chickens and pigs, and these were placed in transport medium similar to that used for humans. These specimens were all kept at 4 °C until transportation. Laboratory testing was conducted by the microbiology laboratory at the Universidad de las Américas.

Phenotypic identification of isolates

All samples from humans and animals were screened by inoculating onto selective CHROMagar COL-APSE chromogenic medium (Paris, France), and they were incubated at 37 °C for 18 hours. Plates were considered positive when at least one typical (coliform) colony had formed (¹⁵15. Haziq M, Momin FA, Bean DC, Hendriksen RS, Haenni M, Phee LM, et al. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens. J Med Microbiol. 2017;66:1554–61.). Subsequently, the typical colonies were inoculated onto BD DIFCO MacConkey Agar medium (Becton Dickinson, Franklin Lakes, New Jersey, USA). Suspected colonies were separated and further confirmed as E. coli or K. pneumoniae through biochemical testing that included inoculation onto Simmons citrate medium, and the triple sugar iron, urea, sulfur indole motility and the methyl red/Voges–Proskauer tests (all media and solutions were from Becton Dickinson); isolates were preserved for future experiments. All isolates were preserved in brain heart infusion broth with 10% glycerol; those used in the study were frozen at −20 °C and those preserved for future research were frozen at −80 °C. ATCC (Manassas, Virginia, USA) strains 25922 for E. coli and 700603 for K. pneumoniae were used for quality control.

Phenotypic resistance to colistin

The isolates of E. coli and K. pneumoniae were incubated in brain heart infusion broth for 18 hours at 37 °C for enrichment. Subsequently, colonies were isolated on nutrient agar and cultivated at 37 °C for 18 hours. These colonies in Muller Hinton broth medium were compared with the 0.5 McFarland standard. Briefly, the broth microdilution test was carried out in line with the recommendations of the Clinical and Laboratory Standards Institute (CLSI standard M100, 2022), in which bacteria diluted to the 0.5 McFarland standard are cultured together with the test antibiotic. Two cut-off points were used: ≤ 2 μg/mL to indicate intermediate resistance and ≥ 4 μg/mL to indicate colistin resistance (CLSI standard M100, 2022). All isolates were then cultured in brain heart infusion broth for cryopreservation for future experiments. The plates were processed in a reader at the recommended wavelength (260 nm). Each isolate was inoculated and subsequently measured in duplicate.

Bacterial DNA extraction

DNA was extracted following the Chelex 100 and proteinase K methods previously described (¹⁶16. Suenaga E, Nakamura H. Evaluation of three methods for effective extraction of DNA from human hair. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;820(1):137–41.). Briefly, all confirmed isolates of E. coli and K. pneumoniae were resuspended in 200 μL of Chelex 10% (Sigma-Aldrich, St. Louis, Missouri, USA) and 5 μL of proteinase K (Invitrogen, Waltham, Massachusetts, USA). Samples were incubated at 56 °C for 60 min. Following this, the samples were vortexed and centrifuged at 8000 g for 2 min. They were then incubated at 96 °C for 20 min. Finally, samples were centrifuged at 8000 g for 5 min and the supernatant was transferred to a clean tube and stored at −20 °C.

Molecular detection of mcr

One-step polymerase chain reaction (PCR) was performed for the mcr-1 gene using the forward primer 5′-GCTACTGATCACCACGCTGT-3′ and the reverse primer 5′-AGCTGAACATACACGGCACA-3′, giving a product size of 698 bp. The QIAGEN PCR kit (QIAGEN, Hilden, Germany) was used. The reaction mix for the PCR contained an amplification mixture of 10 µL, with a concentration of 1 µM of multiplex master mix, 0.2 µM of the mixture of primer pairs for the mcr-1 gene, and 1 µM of Q solution, following the manufacturer’s instructions for multiplex PCR kits (¹⁷17. Yamaguchi T, Kawahara R, Harada K, Teruya S, Nakayama T, Motooka D, et al. The presence of colistin resistance gene mcr-1 and -3 in ESBL producing Escherichia coli isolated from food in Ho Chi Minh City, Vietnam. FEMS Microbiol Lett. 2018;365(11):fny100., ¹⁸18. QIAGEN. QIAGEN multiplex PCR handbook. Hilden (Germany): QIAGEN; 2010.).

PCR was performed in a Mastercycler thermocycler (Eppendorf, Hamburg, Germany) using gradient operation, with the initial denaturation step at 95 °C for 15 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 90 s, extension at 72 °C for 90 s and a final extension at 72 °C for 10 min. PCR products were analyzed using SyBr Safe DNA Gel Stain (Invitrogen) in 2% (w/v) agarose gel in 1X tris–borate–EDTA (ethylenediaminetetraacetic acid) buffer, and electrophoresis was performed at 100 V for 40 min in an Enduro Gel XL Electrophoresis System (Labnet International, Edison, New Jersey, USA). The positive control DNA for the mcr-1 gene was kindly donated by the Osaka Institute of Public Health, Japan. Finally, to confirm the variant of the mcr gene, the 3130 Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA) was used for Sanger sequencing on positive amplification products from PCR. The sequences obtained were aligned with others already described and characterized in the GenBank database using the Geneious 8.0 (Biomatters, Auckland, New Zealand) and MEGA X (Molecular Evolutionary Genetics Analysis, https://www.megasoftware.net/docs) bioinformatics programs. A summary of study procedures is presented in Figure 1.

Sample size calculation

The sample size was calculated based on the formula N = (Z1− α/2)2p(1 − p)/d2, where Z1 − α/2 is the value of normal deviation at a 95% confidence level, p is the expected frequency of colistin resistance in about 50% of farms, according to the reports by Yamamoto et al. (¹²12. Yamamoto Y, Calvopina M, Izurieta R, Villacres I, Kawahara R, Sasaki M, et al. Colistin-resistant Escherichia coli with mcr genes in the livestock of rural small-scale farms in Ecuador. BMC Res Notes. 2019;12(1):121.), with 15% relative precision (d), a design effect of 2 for cluster sampling and a nonresponse rate of 10%.

Cluster random sampling was adopted to select the host from which samples were collected. A total of 20 clusters were selected from the available hamlets. Then 20 households were selected from each cluster by simple random sampling. From each household that agreed to participate, one adult human or one animal was recruited into the study. This process was followed until the desired total sample size of 400 was achieved.

Data entry and statistical analysis

Data were extracted from the study forms and laboratory records, double entered into a Microsoft Excel 2010 spreadsheet (Microsoft, Redmond, Washington, USA) and analyzed using EpiData v. 2.2.3.187 (EpiData, Odense, Denmark). The prevalence of bacterial isolates and their genotypic and phenotypic resistance to colistin were calculated and reported with a 95% confidence interval (CI). Phenotypic resistance was defined as bacterial growth occurring in the broth microdilution test at ≥ 4 μg/mL of colistin and genotypic resistance as the presence of mcr-1. The prevalence of genotypic and phenotypic resistance in relation to geographical location and different hosts was compared using the χ² test. A P value < 0.05 was considered statistically significant.

Ethics

Approval was obtained from the Ethics Committee for the Investigation on Human Beings at Universidad San Francisco de Quito (approval code 2018-110E), in accordance with the Declaration of Helsinki. Ethics approval was also obtained from the Union Ethics Advisory Group (EAG approval number 19/21) of the International Union Against Tuberculosis and Lung Disease, Paris, France. All participants included in the study signed approved informed consent forms.

RESULTS

Prevalence of E. coli and K. pneumoniae

A total of 438 individual fecal samples were obtained from rural hamlets of the coastal and the Amazon regions of Ecuador. These included 226 samples from Santo Domingo province in the coastal region (72 from humans, 78 from pigs, 76 from chickens) and 212 samples from Pastaza province in the Amazon region (65 from humans, 69 from pigs, 78 from chickens).

The overall prevalence of E. coli isolates was 86.3% (378/438; 95% CI: 82.1 to 90.5) and the overall prevalence of K. pneumoniae was 37.4% (164/438; 95% CI: 32.0 to 42.0]. The prevalences of these isolates, stratified by province and by host, are shown in Table 1 and Table 2, respectively. For E. coli isolates, the prevalence was similar in Santo Domingo and in Pastaza among chickens, pigs and humans and ranged between about 85% and 88%. For K. pneumoniae isolates, however, there was a statistically significant higher prevalence in Santo Domingo (42.9%) compared with Pastaza (31.6%) (P < 0.05) and a significantly higher prevalence in humans (72.3%) compared with chickens (27.9%) and with pigs (15.0%) (P < 0.01).

Prevalence of mcr-1 in E. coli and K. pneumoniae

The overall prevalence of mcr-1 in E. coli isolates was 90.0% (340/378; 95% CI: 86.1 to 93.8) and the overall prevalence in K. pneumoniae isolates was 19.5% (32/164; 95% CI:12.1 to 27.9). The prevalences of mcr-1 in E. coli and K. pneumoniae, stratified by province and by host, are also shown in Table 1 and Table 2, respectively. For E. coli isolates, there was a statistically significant higher prevalence in isolates from Santo Domingo (96.5%, 191/198) compared with Pastaza (82.7%, 149/180) (P < 0.001) and a statistically significant higher prevalence in humans (95.6%, 111/116) and chickens (91.8%, 123/134) compared with pigs (82.8%, 106/128) (P < 0.0.5). For K. pneumoniae isolates, the prevalence of mcr-1 was similar in Santo Domingo and Pastaza, at about 19%, and similar between humans, chickens and pigs, at about 16–21%. The GenBank accession number MW527090, corresponding to the phosphoethanolamine–lipid A transferase mcr-1 gene, was obtained from isolate LR37 from a healthy human in Santo Domingo province.

Prevalence of phenotypic resistance to colistin

Two (0.5%) of the 378 E. coli isolates showed statistically significant phenotypic resistance to colistin (Table 3). Two (1.2%) of the 164 K. pneumoniae isolates showed statistically significant phenotypic resistance to colistin, with these two isolates being identified in pigs (Table 4).

Correlation between colistin-resistant phenotypes and mcr-1

Correlations between phenotypic resistance and the presence of mcr-1 for E. coli and K. pneumoniae are shown in Table 3 and Table 4, respectively. For E. coli, mcr-1 was found in all isolates from humans, pigs and chickens showing phenotypic intermediate resistance (≤ 2 µg/mL) to colistin. The mcr-1 gene was also found in the two E. coli isolates showing resistance (≥ 4 µg/mL) to colistin (Table 3). For K. pneumoniae, mcr-1 was found in isolates from humans, pigs and chickens with intermediate resistance to colistin. The mcr-1 gene was not found in the two K. pneumoniae isolates showing resistance to colistin (Table 4).

DISCUSSION

To our knowledge, this is the first study in Ecuador, and one of the first in Latin America, that has assessed the prevalence of fecal carriage of colistin-resistance among E. coli and K. pneumoniae isolates from healthy humans and their backyard animals living in rural areas of two different regions. We found a surprisingly low prevalence of phenotypic resistance to colistin (< 1%), but a high prevalence of molecular resistance in E. coli, with mcr-1 present in 90% of fecal samples with E. coli.

The high prevalence of E. coli in all types of hosts was consistent with previous studies, confirming that this bacterium is one of the most common colonizers of the gastrointestinal tract in both animals and humans (¹⁴14. Bastidas-Caldes C, Ochoa J, Guerrero-Latorre L, Moyota-Tello C, Tapia W, Rey-Pérez JM, et al. Removal of extended-spectrum beta-lactamase-producing Escherichia coli, ST98, in water for human consumption by black ceramic water filters in low-income Ecuadorian highlands. Int J Environ Res Public Health. 2022;19(8):4736.). The overall prevalence of K. pneumoniae in the three types of hosts was lower, at 37%, although humans had a higher prevalence of the organism compared with chickens and pigs. K. pneumoniae naturally colonizes the respiratory and gastrointestinal tracts of humans, and the bacterium is one of the main etiological pathogens of infections with clinical relevance (¹⁹19. Collignon P, Beggs JJ, Walsh TR, Gandra S, Laxminarayan R. Anthropological and socioeconomic factors contributing to global antimicrobial resistance: a univariate and multivariable analysis. Lancet Planet Health. 2018;2(9):e398–405.). The low prevalence of K. pneumoniae in chickens and pigs in our study has been found elsewhere (²⁰20. Franklin-Alming FV, Kaspersen H, Hetland MAK, Bakksjø RJ, Nesse LL, Leangapichart T, et al. Exploring Klebsiella pneumoniae in healthy poultry reveals high genetic diversity, good biofilm-forming abilities and higher prevalence in turkeys than broilers. Front Microbiol. 2021;12:725414.) and might be due to their diet, competition in the environment or genetic virulence factors responsible for the colonization of enteric systems in animals (²¹21. Young T, Bray A, Nagpal R, Caudell D, Yadav H, Zafar M. Animal model to study Klebsiella pneumoniae gastrointestinal colonization and host-to-host transmission. Infect Immun. 2020;88(11):e00071-20., ²²22. Cao L, Li X, Xu Y, Shen J. Prevalence and molecular characteristics of mcr-1 colistin resistance in Escherichia coli: isolates of clinical infection from a Chinese university hospital. Infect Drug Resist. 2018;11:1597–603.).

There was a surprisingly low prevalence of phenotypic resistance to colistin in our study. Our results concur with a recent review that found a global prevalence of colistin resistance of 1.6%, with incidence rates rising during the past 5 years on all continents, largely as a result of inappropriate use of the antibiotic in animal husbandry (²³23. Binsker U, Käsbohrer A, Hammerl JA. Global colistin use: a review of the emergence of resistant Enterobacterales and the impact on their genetic basis. FEMS Microbiol Rev. 2022;46(1):fuab049.). We used standard laboratory methodology (broth microdilution) for determining the minimum inhibitory concentration values for colistin, so we believe our results are accurate. Of note, however, when laboratory susceptibility testing of our samples was conducted in 2020 and 2021, the CLSI break point of ≤ 2 µg/mL of colistin was regarded as indicating susceptibility. The 2022 version of the CLSI standard indicates that bacterial growth at that concentration should be considered as indicating intermediate resistance, which is how we have presented the data.

A high prevalence of mcr-1 (90%) was found in the E. coli isolates, and this aligns with the rapid worldwide spread of this gene since it was first reported in 2016 in China. There are several variants of the mcr gene, but mcr-1 appears to be the predominant variant and has spread at a rate that is 95% faster than the other 9 variants (mcr-2 to mcr-10) (³3. Elbediwi M, Li Y, Paudyal N, Pan H, Li X, Xie S, et al. Global burden of colistin-resistant bacteria: mobilized colistin resistance genes study (1980–2018). Microorganisms. 2019;7(10):1–18., ⁸8. Bastidas-Caldes C, de Waard JH, Salgado MS, Villacís MJ, Coral-Almeida M, Yamamoto Y, et al. Worldwide prevalence of mcr-mediated colistin-resistance Escherichia coli in isolates of clinical samples, healthy humans, and livestock—a systematic review and meta-analysis. Pathogens. 2022;11(6):659.). The mcr-1 gene is certainly the most common in Latin America and the Caribbean, and the country with the highest prevalence is Brazil, with nearly 45% of E. coli isolates testing positive for the gene (²⁴24. 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):pii=30214.).

We found a higher prevalence of mcr-1 in E. coli isolates from the coastal region compared with the Amazon region, and in humans and chickens compared with pigs. Other studies in Ecuador have also found differing prevalence rates, depending on the source. For example, a low prevalence was found in dogs, at 25% (¹¹11. Albán MV, Núñez EJ, Zurita J, Villacís JE, Tamayo R, Sevillano G, et al. Canines with different pathologies as carriers of diverse lineages of Escherichia coli harbouring mcr-1 and clinically relevant β-lactamases in central Ecuador. J Glob Antimicrob Resist. 2020;22:182–3.), and irrigation water, at 18% (¹³13. Montero L, Irazabal J, Cardenas P, Graham JP, Trueba G. Extended-spectrum beta-lactamase producing-Escherichia coli isolated from irrigation waters and produce in Ecuador. Front Microbiol. 2021;12:709418.), whereas in chickens rates of more than 95% were found (¹²12. Yamamoto Y, Calvopina M, Izurieta R, Villacres I, Kawahara R, Sasaki M, et al. Colistin-resistant Escherichia coli with mcr genes in the livestock of rural small-scale farms in Ecuador. BMC Res Notes. 2019;12(1):121., ²⁵25. Hmede Z, Kassem II. The colistin resistance gene mcr-1 is prevalent in commensal Escherichia coli isolated from preharvest poultry in Lebanon. Antimicrob Agents Chemother. 2018;62(11):e01304-18.). We do not have a clear explanation for why there are differences, but diet, animal husbandry practices and the environment might play a part.

In K. pneumoniae isolates, we found a lower prevalence of mcr-1, at less than 20%, with little difference between geographical regions and type of host. However, this prevalence in our study was higher than those found elsewhere: in Taiwan, 5.5% of chickens and 0.4% of pigs were reported to harbor mcr-1 (²⁶26. Yang YQ, Li YX, Lei CW, Zhang AY, Wang HN. Novel plasmid-mediated colistin resistance gene mcr-7.1 in Klebsiella pneumoniae. J Antimicrob Chemother. 2018;73(7):1791–5., ²⁷27. Lai CC, Lin YT, Lin YT, Lu MC, Shi ZY, Chen YS, et al. Clinical characteristics of patients with bacteraemia due to the emergence of mcr-1-harbouring Enterobacteriaceae in humans and pigs in Taiwan. Int J Antimicrob Agents. 2018 Nov 1;52(5):651–7.), while in China 11% of chickens and 7% of pigs with resistant K. pneumoniae isolates harbored the gene (²⁸28. Wang X, Wang Y, Zhou Y, Li J, Yin W, Wang S, et al. Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae. Emerg Microbes Infect. 2018;7(1):122.).

There was no correlation between phenotypic resistance and the presence of mcr-1. This finding has been previously described by others (²⁹29. Pillonetto M, Mazzetti A, Becker GN, Siebra CA, Arend LNVS, Barth AL. Low level of polymyxin resistance among nonclonal mcr-1–positive Escherichia coli from human sources in Brazil. Diagn Microbiol Infect Dis. 2019;93(2):140–2., ³⁰30. Lentz SA, de Lima-Morales D, Cuppertino VM, Nunes LDS, da Motta AS, Zavascki AP, et al. Escherichia coli harbouring mcr-1 gene isolated from poultry not exposed to polymyxins in Brazil. Euro Surveill. 2016;21(26):pii=30267.), and it may be explained by the multifactorial aspects of antimicrobial resistance, such as (i) the diversity of plasmid types carrying mcr-1 (³¹31. Wu R, Yi LX, Yu LF, Wang J, Liu Y, Chen X, et al. Fitness advantage of mcr-1-bearing IncI2 and IncX4 plasmids in vitro. Front Microbiol. 2018;9:331.), (ii) strong or weak plasmid promotors influencing the expression of the genes (³²32. Lu S, Li D, Wang L, Bi Y, Wang M, Yang F. Promoter variations associated with expression of mcr-1 gene and level of colistin resistance. Int J Antimicrob Agents. 2021;58(2):106371.), and (iii) the fusion plasmid phenomenon that increases the transfer capabilities of mcr genes between hosts (⁹9. Hoa HTT, Higashi A, Yamaguchi T, Kawahara R, Calvopina M, Bastidas-Caldés A, et al. Fusion plasmid carrying the colistin resistance gene mcr of Escherichia coli isolated from healthy residents. J Glob Antimicrob Resis. 2022;30:152–4.). Only four bacterial isolates were resistant, and all were found in pigs. The two resistant E. coli isolates were positive for mcr-1, indicating plasmid-induced resistance, while the two resistant K. pneumoniae isolates were negative for mcr-1, presumably indicating resistance due to chromosomal mutations. The high prevalence mcr-1 in E. coli with intermediate resistance, however, is cause for concern, as this might lead to further full-blown resistance if the misuse of colistin continues.

Strengths and limitations

The strengths of this study were the large sample size, which exceeded our estimated sample size; a robust system for collecting specimens; sound laboratory techniques that followed international guidelines; and conducting and reporting the study in line with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines (³³33. Von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbrouckef JP. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Bull World Health Organ. 2007;85(11):867–72.). A further strength was the implementation of the study in two different regions and among rural settlements, together with the inclusion of humans and two types of backyard animals.

However, there were some limitations. It was difficult to collect samples consistently during the study period due to the COVID-19 pandemic, and we had several refusals to participate from households in the settlements. Genetic and epidemiological studies are needed to compare these findings and determine whether there are other strains in the country. Furthermore, studies on the expression of the mcr-1 gene are necessary to understand better the relationship between molecular and phenotypic resistance because this is dynamic and multifactorial for colistin. The clonal distribution between humans, chickens and pigs, and the presence of the mcr-1 gene in plasmids were not determined, and these are areas to pursue in further research. Finally, we did not collect any information about colistin use in animals in the settlements that we studied.

Conclusions

The prevalence of colistin resistance in bacterial isolates of E. coli and K. pneumoniae was evaluated from fecal samples from healthy humans and their backyard animals living in rural Ecuador.

The prevalence of molecular resistance to colistin (identified by the presence of mcr-1) was high, especially in E. coli isolates (90%). However, the low prevalence of phenotypic resistance associated with these strains was striking (< 1%). In K. pneumoniae isolates, phenotypic resistance occurred independently of the presence of mcr-1 (1.2%), with the chromosomal mechanism of colistin resistance probably being the main factor involved. These findings raise important concerns about the potential spread of drug-resistant pathogens in the community, which will add to the overall burden of antimicrobial resistance unless firm action is taken.

To counter this, we make two important recommendations. First, our study methodology should serve as a basis for ongoing surveillance of colistin resistance in three species of hosts, or carriers, and this should be replicated in sentinel sites all over the country. Second, there is an urgent need to regulate the use of colistin in animal husbandry and to avoid its overuse in human clinical cases for which there is no clear indication. In January 2020, Ecuador banned the veterinary use of colistin, and it has since been shown that the sales of colistin as a growth promoter have decreased (³⁴34. Butzin-Dozier Z, Waters WF, Baca M, Vinueza RL, Saraiva-Garcia C, Graham J. Assessing upstream determinants of antibiotic use in small-scale food animal production through a simulated client method. Antibiotics (Basel). 2021;10(1):2.). An encouraging report from China showed that banning colistin as an animal growth promoter led to substantial decreases in colistin-resistant E. coli in pigs, chickens and humans (³⁵35. Wang Y, Xu C, Zhang R, Chen Y, Shen Y, Hu F, et al. Changes in colistin resistance and mcr-1 abundance in Escherichia coli of animal and human origins following the ban of colistin-positive additives in China: an epidemiological comparative study. Lancet Infect Dis. 2020;20(10):1161–71.). Therefore, regulatory policy combined with continual colistin monitoring can work and will help prevent the spread of colistin resistance in Ecuador and elsewhere.

Disclaimer.

Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the Revista Panamericana de Salud Pública/Pan American Journal of Public Health or those of the Pan American Health Organization.

Acknowledgements.

This research study was developed through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases (TDR), the United Nations Children’s Fund (UNICEF), the United Nations Development Programme (UNDP) and the World Bank; it is hosted at the World Health Organization. The specific SORT IT program that led to this study included an implementation partnership between TDR and the Pan American Health Organization, the WHO country offices of Colombia and Ecuador; the Ministry of Health and Social Protection, Colombia; the Food and Agriculture Organization, Sierra Leone; Sustainable Health Systems, Freetown, Sierra Leone; the Tuberculosis Research and Prevention Center, nongovernmental organization, Armenia; the International Union Against Tuberculosis and Lung Disease, France and India offices (South-East Asia); Institute of Tropical Medicine, Antwerp, Belgium; Damien Foundation, Belgium; Indian Council of Medical Research–National Institute of Epidemiology; Jawaharlal Institute of Postgraduate Medical Education and Research; GMERS Medical College, Gotri, India; India Medical College Baroda; Sri Manakula Vinayagar Medical College, India; Public Health Ontario, Canada; Universidade Federal de Ciências de Saúde de Porto Alegre, Brazil; Universidade de Brasilia, Brazil; Universidad de Concepción, Chile; Universidad de los Andes, Colombia; Universidad Pontificia Bolivariana, Colombia; Universidad Pedagógica y Tecnológica de Colombia; Universidad Central del Ecuador; California State University, Fullerton, California, USA; and Universidad Autónoma de Yucatán, México.

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