The Plasmid-Mediated Kluyvera-Like arnBCADTEF Operon Confers Colistin (Hetero)Resistance to Escherichia coli

ABSTRACT The use of colistin as a last-resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB system, or still unknown mechanisms. This work analyzed 74 Escherichia coli isolates from healthy swine, turkey, or bovine, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found, among which 45% carried this gene on highly mobile plasmids, followed by 4 strains lacking previously known resistance determinants or 2 with mcr-4 (1 in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to strains with mutations that confer colistin resistance. The expression of mcr genes negatively controls the transcription of the arnBCADTEF operon itself, a downregulation that was also observed in the four isolates lacking known resistance determinants, with three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed an IncFII plasmid of 62.1 kb carrying an extra copy of the arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, called pArnT1, was cured by ethidium bromide, and the cells lost resistance to colistin in parallel. Furthermore, a susceptible E. coli strain acquired heteroresistance after transformation with pArnT1 or pBAD24 carrying the Kluyvera-like arnBCADTEF operon, revealing it as a new colistin resistance determinant.

Mobilization of a Kluyvera-like arnBCADTEF operon, besides mcr genes, confers colistin resistance to Escherichia coli isolated from healthy animals

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB two-component system and also by some other(s) still unknown mechanism(s). This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine animals, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to control strains carrying mutations that confer colistin resistance. The expression of mcr genes negatively controls arnBCADTEF expression, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 60 Kb encoding an arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, named pArnT1, was cured by ethidum bromide and lost in parallel to colistin resistance. This work reveals that, besides mcr genes and chromosomal mutations, mobilization of arnBCADTEF operon represents a colistin resistance mechanism whose spread and relevance for public health should be carefully surveyed.Abstract ImportanceColistin is an old antibiotic that has returned to first-line fighting against (Gram negative) microorganisms after pandemic rising of antimicrobial resistance. However, low susceptibility to colistin is also becoming spread, mainly by plasmid mobilization of one of the enzymes (encoded by mcr genes) that modify covalently the external layer (the lipid A component of the lipopolysaccharide) of bacterial envelope, interfering antibiotic effectiveness. The second enzymatic system that performs envelope modification and confers colistin resistance when overexpressed is encoded by arnBCADTEF operon, a set of seven genes with location restricted (up to now) to the chromosome of Gram negative bacteria. This work describes plasmid mobilization of this operon between enterobacteria, from Kluyvera to Escherichia coli, where a Kluyvera-like arnBCADTEF operon carried by pArnT1 might represent, besides mcr genes, a potential risk for antimicrobial therapy and might require careful surveillance.

Microbiota oral e retal de calitriquídeos (Callithrix sp.) em área antropizada de Mata Atlântica, Rio de Janeiro, Brasil

RESUMO A proximidade dos primatas não humanos (PNH) com o ser humano pode ser considerada um fator de risco para transmissão de bactérias entre essas duas populações. Neste estudo, foi investigada a microbiota anfibiôntica aeróbica oral e retal de calitriquídeos em um fragmento de Mata Atlântica localizado no Rio de Janeiro, Brasil, e foram realizados testes fenotípicos para detecção de bactérias multirresistentes nos isolados encontrados. Foram capturados 14 calitriquídeos e coletadas 21 amostras (14 de cavidade oral e sete de cavidade retal) em dois pontos da mata próximos às habitações humanas. As espécies mais frequentes, na cavidade oral, foram Klebsiella oxytoca (50,0%), K. pneumoniae (28,6%), Kluyvera ascorbata (21,4%) e Stenotrophomonas maltophilia (21,4%) e, na cavidade retal, K. pneumoniae (85,7%), Escherichia coli (28,6%) e Enterobacter spp. (42,9%). Todos os 48 isolados da família Enterobacteriaceae foram negativos para ESBL (betalactamase de espectro ampliado), mostrando-se não produtores da enzima nos dois métodos utilizados: disco-aproximação e método de detecção automatizado. Na pesquisa de ERC (enterobactérias resistentes a carbapenêmicos), esses mesmos isolados não apresentaram resistência aos antibióticos imipenem, meropenem e ertapenem. Todas as bactérias isoladas apresentam um potencial zoonótico, o que representa um risco à saúde pública e à conservação das espécies.

INFECCION URINARIA POR Kluyvera ascorbata: REPORTE DE UN CASO

La bacteria Kluyvera ascorbata forma parte de la microbiota normal del tubo digestivo humano; pero en ocasiones, puede ocasionar infecciones potencialmente graves. Se presenta el caso de una paciente con pancreatitis aguda que luego de un antecedente de hospitalización y de portar sonda vesical, reingresa al hospital por una reagudización de su enfermedad acompañada de molestias urinarias, aislándose en urocultivo a Kluyvera ascorbata multirresistente. Este cuadro clínico pudo deberse a una infección intrahospitalaria relacionada al uso de sonda vesical; además, existe la posibilidad de que la paciente desarrollara una bacteriemia asociada a Kluyvera ascorbata. PALABRAS CLAVE: Kluyvera, Infecciones Urinarias, Pancreatitis (DeCS)