Simple Phenotypic Tests To Improve Accuracy in Screening Chromosomal and Plasmid-Mediated Colistin Resistance in Gram-Negative Bacilli.

CLSI and EUCAST recommend that only broth microdilution (BMD) should be used for routine colistin susceptibility testing; however, this technique can be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of a colistin agar spot test (COL-AS) and a colistin drop test (COL-DT) compared to BMD. COL-AS and COL-DT were assessed with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp., and 39 Pseudomonas aeruginosa For COL-AS, 3.0 µg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm2 spot. For COL-DT, 10 µl of a 16 µg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of the tested strain (0.5 McFarland standard). Colistin solution was made either by dissolving powder or by disk elution in cation-adjusted Mueller-Hinton broth (CA-MHB). Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 strains with MICs that fluctuated from 2 to 4 µg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in Gram-negative bacilli.

Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: A retrospective study, 2012-2018.

OBJECTIVE: To describe the resistance profile and the genetic characteristics of Escherichia coli isolates that harbor the mobilizable colistin resistance gene mcr-1 in Argentina. METHODS: This was a retrospective study of 192 E. coli isolates positive for mcr-1 obtained from 69 hospitals of Buenos Aires City and 14 Argentinean provinces in 2012 - 2018. The antimicrobial susceptibility was performed by agar diffusion, broth macrodilution, and/or agar dilution. Standard polymerase chain reaction (PCR) was performed to detect resistance genes and incompatibility groups; specific PCR was applied to discriminate between blaCTX-M allelic groups and mcr-1.5 variant. The genetic relatedness among isolates was evaluated by XbaI-pulsed field gel electrophoresis and multilocus sequence typing in a subset of isolates. RESULTS: All E. coli isolates showed minimal inhibitory concentrations to colistin ≥ 4µg/mL; nearly 50% were resistant to third-generation cephalosporins, with CTX-M-2 being the main extended-spectrum ß-lactamase detected. Five E. coli were carbapenemase-producers (3 NDM, 2 KPC). The mcr-1.5 variant was detected in 13.5% of the isolates. No genetic relationship was observed among the mcr-1-positive E. coli clinical isolates, but a high proportion (164/192; 85.4%) of IncI2 plasmids was detected. CONCLUSIONS: The presence of IncI2 plasmids among highly diverse E. coli clones suggests that the mcr-1 gene's wide distribution in Argentina may be driven by the horizontal transmission of IncI2 plasmids.

Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: A retrospective study, 2012–2018

[ABSTRACT]. Objective. To describe the resistance profile and the genetic characteristics of Escherichia coli isolates that harbor the mobilizable colistin resistance gene mcr-1 in Argentina. Methods. This was a retrospective study of 192 E. coli isolates positive for mcr-1 obtained from 69 hospitals of Buenos Aires City and 14 Argentinean provinces in 2012 – 2018. The antimicrobial susceptibility was performed by agar diffusion, broth macrodilution, and/or agar dilution. Standard polymerase chain reaction (PCR) was performed to detect resistance genes and incompatibility groups; specific PCR was applied to discriminate between blaCTX-M allelic groups and mcr-1.5 variant. The genetic relatedness among isolates was evaluated by XbaI-pulsed field gel electrophoresis and multilocus sequence typing in a subset of isolates. Results. All E. coli isolates showed minimal inhibitory concentrations to colistin ≥ 4μg/mL; nearly 50% were resistant to third-generation cephalosporins, with CTX-M-2 being the main extended-spectrum β-lactamase detected. Five E. coli were carbapenemase-producers (3 NDM, 2 KPC). The mcr-1.5 variant was detected in 13.5% of the isolates. No genetic relationship was observed among the mcr-1-positive E. coli clinical isolates, but a high proportion (164/192; 85.4%) of IncI2 plasmids was detected. Conclusions. The presence of IncI2 plasmids among highly diverse E. coli clones suggests that the mcr-1 gene’s wide distribution in Argentina may be driven by the horizontal transmission of IncI2 plasmids.

[RESUMEN]. Objetivo. Describir el perfil de resistencia y las características genéticas de aislamientos clínicos de Escherichia coli que portan el gen movilizable de resistencia a colistina mcr-1 en Argentina. Métodos. Se realizó un estudio retrospectivo para analizar 192 aislamientos de E. coli mcr-1 positivo, obtenidos en 69 hospitales de la Ciudad de Buenos Aires y 14 provincias de Argentina entre 2012 y 2018. La sensibilidad a los antimicrobianos se analizó mediante los métodos de difusión en agar, macrodilución en caldo y/o dilución en agar. Se aplicó la técnica estándar de reacción en cadena de la polimerasa (PCR) para detectar genes de resistencia y grupos de incompatibilidad; se aplicó PCR específica para distinguir entre variantes alélicas del gen blaCTX-M y la variante mcr-1.5. La relación genética entre los aislamientos fue evaluada mediante la técnica de electroforesis en gel de campo pulsado usando la enzima Xbal y la tipificación por secuencias de múltiples locus en un subconjunto de aislamientos. Resultados. Todos los aislamientos de E. coli mostraron concentraciones inhibitorias mínimas de colistina ≥ 4μg/mL. Casi el 50% mostró resistencia a las cefalosporinas de tercera generación y CTX-M-2 fue la β-lactamasa de espectro extendido que más se detectó. Cinco aislamientos de E. coli mostraron ser productoras de carbapenemasas (3 NDM, 2 KPC). La variante mcr-1.5 se detectó en 13,5% de las cepas aisladas. No se observó relación genética entre los aislamientos clínicos estudiados de E. coli positivas para mcr-1, aunque sí se detectó una proporción elevada (164/192; 85,4%) de plásmidos Incl2. Conclusiones. La elevada ocurrencia de plásmidos IncI2 en un grupo altamente diverso de clones de E. coli podría indicar que la amplia difusión del gen mcr-1 en Argentina estaría asociada a la transmisión horizontal de plásmidos IncI2.

[RESUMO]. Objetivo. Descrever o perfil de resistência e as características genéticas de isolados clínicos de Escherichia coli que carregam o gene mobilizábel de resistência à colistina mcr-1 na Argentina. Métodos. Neste estudo retrospectivo, foram analizados 192 isolados de E. coli positivos para mcr-1 obtidos em 69 hospitais da Cidade de Buenos Aires e 14 províncias da Argentina, entre 2012 e 2018. A sensibilidade aos antimicrobianos foi examinada usando métodos de difusão em ágar, macrodiluição em caldo e/ou diluição em ágar. A técnica padrão de reação em cadeia da polimerase (PCR) foi aplicada para detectar genes de resistência e grupos de incompatibilidade; a PCR específica foi aplicada para discriminar entre variantes alélicas do gene blaCTX-M e a variante mcr-1.5. A relação genética entre os isolados foi avaliada por eletroforese em gel de campo pulsado usando a enzima XbaI e a tipagem por sequências de múltiplos lócus, em um subconjunto de isolados. Resultados. Todos os isolados de E. coli apresentaram concentrações inibitórias mínimas de colistina ≥4μg/ mL. Quase 50% foram resistentes às cefalosporinas de terceira geração, e CTX-M-2 foi a β-lactamase de espectro estendido mais detectada. Cinco isolados de E. coli foram produtores de carbapenemase (3 NDM, 2 KPC). A variante mcr-1.5 foi detectada em 13,5% dos isolados. Não foi observada relação genética entre os isolados clínicos de E. coli positivos para mcr-1, mas foi detectada uma alta proporção (164/192; 85,4%) de plasmídeos IncI2. Conclusões. A alta ocorrência de plasmídeos IncI2 em um grupo altamente diverso de clones de E. coli sugere que a ampla distribuição do gene mcr-1 na Argentina estaria associada a transmissão horizontal de plasmídeos IncI2.