Integrating Scalable Genome Sequencing Into Microbiology Laboratories for Routine Antimicrobial Resistance Surveillance.

Antimicrobial resistance (AMR) is considered a global threat, and novel drug discovery needs to be complemented with systematic and standardized epidemiological surveillance. Surveillance data are currently generated using phenotypic characterization. However, due to poor scalability, this approach does little for true epidemiological investigations. There is a strong case for whole-genome sequencing (WGS) to enhance the phenotypic data. To establish global AMR surveillance using WGS, we developed a laboratory implementation approach that we applied within the NIHR Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance. In this paper, we outline the laboratory implementation at 4 units: Colombia, India, Nigeria, and the Philippines. The journey to embedding WGS capacity was split into 4 phases: Assessment, Assembly, Optimization, and Reassessment. We show that on-boarding WGS capabilities can greatly enhance the real-time processing power within regional and national AMR surveillance initiatives, despite the high initial investment in laboratory infrastructure and maintenance. Countries looking to introduce WGS as a surveillance tool could begin by sequencing select Global Antimicrobial Resistance Surveillance System (GLASS) priority pathogens that can demonstrate the standardization and impact genome sequencing has in tackling AMR.

Complexity of Genomic Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Isolates in Colombia Urges the Reinforcement of Whole Genome Sequencing-Based Surveillance Programs.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging public health problem. This study explores the specifics of CRKP epidemiology in Colombia based on whole genome sequencing (WGS) of the National Reference Laboratory at Instituto Nacional de Salud (INS)'s 2013-2017 sample collection. METHODS: A total of 425 CRKP isolates from 21 departments were analyzed by HiSeq-X10®Illumina high-throughput sequencing. Bioinformatic analysis was performed, primarily using the pipelines developed collaboratively by the National Institute for Health Research Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance (AMR), and AGROSAVIA. RESULTS: Of the 425 CRKP isolates, 91.5% were carbapenemase-producing strains. The data support a recent expansion and the endemicity of CRKP in Colombia with the circulation of 7 high-risk clones, the most frequent being CG258 (48.39% of isolates). We identified genes encoding carbapenemases blaKPC-3, blaKPC-2, blaNDM-1, blaNDM-9, blaVIM-2, blaVIM-4, and blaVIM-24, and various mobile genetic elements (MGE). The virulence of CRKP isolates was low, but colibactin (clb3) was present in 25.2% of isolates, and a hypervirulent CRKP clone (CG380) was reported for the first time in Colombia. ST258, ST512, and ST4851 were characterized by low levels of diversity in the core genome (ANI > 99.9%). CONCLUSIONS: The study outlines complex CRKP epidemiology in Colombia. CG258 expanded clonally and carries specific carbapenemases in specific MGEs, while the other high-risk clones (CG147, CG307, and CG152) present a more diverse complement of carbapenemases. The specifics of the Colombian situation stress the importance of WGS-based surveillance to monitor evolutionary trends of sequence types (STs), MGE, and resistance and virulence genes.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016.

Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1-positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1, including 8 Escherichia coli, 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate. E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10-5 to 2.07 × 10-1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids.

An outbreak of Salmonella enterica subsp. enterica serovar Give associated with foodborne illness in the department of Vichada, Colombia, 2015 / Brote de Salmonella enterica subsp. enterica serovar Give asociado con enfermedad transmitida por alimentos en Vichada, Colombia, 2015.

Introduction: Salmonella enterica subsp. enterica serovar Give is found in ruminants, pigs, poultry, and aquatic environments, but rarely in humans. In Colombia, this serotype was ranked 11th. in the laboratory surveillance of acute diarrheal disease between 2000 and 2013. Objective: To characterize phenotypic and genotypic isolates of Salmonella related to an outbreak of foodborne Illness in the department Vichada in the fifth epidemiological week of 2015. Materials and methods: Following the Instituto Nacional de Salud method, we tested 37 fecal samples for Salmonella spp. while the sample of canned sardines was processed according to the ISO 6579:2002 Cor.1:2004 standard. The isolates were confirmed by serology and/or real-time PCR, antimicrobial susceptibility tests, and pulsed-field gel electrophoresis with the XbaI and BlnI enzymes. Results: All human isolates (11) and that from food (1) were identified as S. Give. The food isolate exhibited tetracycline resistance. PFGE analysis with XbaI grouped ten isolates from samples of human origin in pattern COIN15JEXX01.0005 and the remaining isolates in COIN15JEXX01.0006 with 96.3% similarity. All isolates were confirmed with the BlnI enzyme, and four (three human isolates and the one from food) were matched to the pattern COIN15JEXA26.002 with 95.65% similarity. Conclusion: Our study confirmed that canned sardines were related to the transmission of S. Give in the outbreak, which is the third one caused by this serotype in Colombia.

Introducción. Salmonella enterica subsp. enterica serovar Give se encuentra en mamíferos rumiantes, cerdos, aves y ambientes acuáticos, pero rara vez en humanos. En Colombia este serotipo ocupó el decimoprimer lugar en frecuencia en la vigilancia por laboratorio de la enfermedad diarreica aguda entre el 2000 y el 2013. Objetivo. Caracterizar el fenotipo y el genotipo de S. Give en aislamientos relacionados con un brote de enfermedad transmitida por alimentos en el departamento de Vichada en la quinta semana epidemiológica del 2015. Materiales y métodos. Se buscó Salmonella spp. en 37 muestras de materia fecal con el método de estudio del Instituto Nacional de Salud. La muestra de sardinas enlatadas fue procesada según la norma ISO6579:2002 Cor.1:2004. Se determinó el serotipo en los aislamientos confirmados mediante serología o PCR en tiempo real, y se hicieron pruebas de sensibilidad a antimicrobianos y electroforesis en gel de campo pulsado con las enzimas Xbal y BlnI. Resultados. Todos los aislamientos de origen humano (11) y el aislamiento del alimento (1), se identificaron como S. Give y este último presentó resistencia a la tetraciclina. El análisis por PFGE-XbaI agrupó bajo el patrón COIN15JEXX01.0005 diez aislamientos de origen humano y a los restantes bajo el COIN15JEXX01.0006, con un 96,3 % de similitud. Los resultados de todos los aislamientos se confirmaron con la enzima BlnI; cuatro de ellos (tres humanos y el del alimento) se agruparon bajo el patrón COIN15JEXA26.002, con un porcentaje de similitud del 95,65 %. Conclusión. El estudio confirmó que las sardinas enlatadas se relacionaron con la transmisión de S. Give en el brote, que es el tercero ocasionado por este serotipo en Colombia.

Brote de Salmonella entérica subsp. entérica serovar Give asociado con enfermedad transmitida por alimentos en Vichada, Colombia, 2015 / An outbreak of Salmonella entérica subsp. entérica serovar Give associated with foodborne illness in the department of Vichada, Colombia, 2015

Resumen | Introducción. Salmonella entérica subsp. entérica serovar Give se encuentra en mamíferos rumiantes, cerdos, aves y ambientes acuáticos, pero rara vez en humanos. En Colombia este serotipo ocupó el decimoprimer lugar en frecuencia en la vigilancia por laboratorio de la enfermedad diarreica aguda entre el 2000 y el 2013. Objetivo. Caracterizar el fenotipo y el genotipo de S. Give en aislamientos relacionados con un brote de enfermedad transmitida por alimentos en el departamento de Vichada en la quinta semana epidemiológica del 2015. Materiales y métodos. Se buscó Salmonella spp. en 37 muestras de materia fecal con el método de estudio del Instituto Nacional de Salud. La muestra de sardinas enlatadas fue procesada según la norma ISO6579:2002 Cor.1:2004. Se determinó el serotipo en los aislamientos confirmados mediante serología o PCR en tiempo real, y se hicieron pruebas de sensibilidad a antimicrobianos y electroforesis en gel de campo pulsado con las enzimas Xbal y BlnI. Resultados. Todos los aislamientos de origen humano (11) y el aislamiento del alimento (1), se identificaron como S. Give y este último presentó resistencia a la tetraciclina. El análisis por PFGE-XbaI agrupó bajo el patrón COIN15JEXX01.0005 diez aislamientos de origen humano y a los restantes bajo el COIN15JEXX01.0006, con un 96,3 % de similitud. Los resultados de todos los aislamientos se confirmaron con la enzima BlnI; cuatro de ellos (tres humanos y el del alimento) se agruparon bajo el patrón COIN15JEXA26.002, con un porcentaje de similitud del 95,65 %. Conclusión. El estudio confirmó que las sardinas enlatadas se relacionaron con la transmisión de S. Give en el brote, que es el tercero ocasionado por este serotipo en Colombia.

Abstract | Introduction: Salmonella enterica subsp. enterica serovar Give is found in ruminants, pigs, poultry, and aquatic environments, but rarely in humans. In Colombia, this serotype was ranked 11th. in the laboratory surveillance of acute diarrheal disease between 2000 and 2013. Objective: To characterize phenotypic and genotypic isolates of Salmonella related to an outbreak of foodborne Illness in the department Vichada in the fifth epidemiological week of 2015. Materials and methods: Following the Instituto Nacional de Salud method, we tested 37 fecal samples for Salmonella spp. while the sample of canned sardines was processed according to the ISO 6579:2002 Cor.1:2004 standard. The isolates were confirmed by serology and/or real-time PCR, antimicrobial susceptibility tests, and pulsed-field gel electrophoresis with the XbaI and BlnI enzymes. Results: All human isolates (11) and that from food (1) were identified as S. Give. The food isolate exhibited tetracycline resistance. PFGE analysis with XbaI grouped ten isolates from samples of human origin in pattern COIN15JEXX01.0005 and the remaining isolates in COIN15JEXX01.0006 with 96.3% similarity. All isolates were confirmed with the BlnI enzyme, and four (three human isolates and the one from food) were matched to the pattern COIN15JEXA26.002 with 95.65% similarity. Conclusion: Our study confirmed that canned sardines were related to the transmission of S. Give in the outbreak, which is the third one caused by this serotype in Colombia.

Capacidad bactericida de Bifidobacterium sp. aislada de leche materna y de heces de neonatos, frente a los principales causantes de enfermedades transmitidas por alimentos / Antibiotic activity of Bifidobacterium sp. isolated from breast milk and newborn faeces, against the main causes for foodborne illnesses

Introducción. La microbiota del tubo digestivo humano contiene bacterias benéficas para la salud que regulan el funcionamiento del colon e inhiben algunos microorganismos patógenos intestinales. Las bifidobacterias aisladas de neonatos y de leche materna se usan como microorganismos probióticos para prevenir enfermedades infecciosas, incluidas las transmitidas por alimentos. Objetivo. Aislar e identificar Bifidobacterium sp. en humanos y determinar su capacidad bactericida frente a patógenos causantes de enfermedades transmitidas por alimentos, importantes en Colombia y en el mundo. Materiales y métodos. Se recolectaron 17 muestras de leche materna, y 19 muestras de meconio y heces de neonatos, en diferentes hospitales de Bogotá. Los 26 aislamientos sospechosos se identificaron a nivel de género mediante PCR 16-23S; para la identificación de especie, se secuenciaron algunos de los aislamientos. La capacidad antagonista de las 26 cepas de Bifidobacterium sp. fue evaluada contra Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644 y E. coli O157:H7 ATCC 35150. Para las cepas que presentaron mayor actividad antagonista, se analizó el extracto inhibidor con ensayos de difusión en placa. Resultados. Todas las cepas amplificaron la banda esperada para la confirmación de género; asimismo, las cepas Bif 013 y Bif 023 se identificaron por secuenciación como Bifidobacterium breve, con una homología del 97%. Del total de cepas, 17 mostraron capacidad de inhibir, al menos, uno de los patógenos evaluados. E. coli ATCC 25922 fue el patógeno más inhibido. Se determinó que la cepa Bif 023 es eficiente como antagonista, ya que inhibió todos los patógenos evaluados. Los halos de inhibición presentaron diámetros mayores a lo esperado, lo que indica una muy buena capacidad antagonista de las cepas nativas. Se aisló Bifidobacterium sp. de leche materna, meconio y heces de neonatos, por lo cual se confirmó que este microorganismo es microbiota humana (2). Conclusión. Se concluye que, debido a la gran capacidad antagonista de la mayoría de las bacterias aisladas, éstas pueden estar cumpliendo una importante función protectora en el recién nacido, en particular las cepas Bif 013 y Bif 023 aisladas de materia fecal. Estos microorganismos deben continuar siendo estudiados para definir su potencial probiótico. También, se pueden evaluar para bioconservación en la industria y contra patógenos transmitidos por alimentos.

Introduction. The microbiota in the human gastrointestinal tract contains beneficial microorganisms for human health, which contribute to the regulation of colonic function and inhibition of some intestinal pathogens growth. Bifidobacterium sp. isolated from newborns and breast milk are used as probiotic microorganisms, which are useful in the prevention of infectious diseases including foodborne illnesses. Objective: To isolate and identify human Bifidobacterium sp. and to determine its antibiotic activity against important pathogens which cause foodborne illnesses in Colombia and the world. Materials and methods. 17 breast milk samples and 19 meconium and newborn faeces samples were collected from different hospitals in Bogotá. 26 presumptive Bifidobacterium strains were identified at genus level by PCR 16-23S; some strains were identified at species level by nucleic acid sequencing. The antagonistic activity of 26 Bifidobacterium sp. strains was tested against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 7644 and E. coli O157:H7 ATCC 35150. For the strains that showed a greater antibiotic activity, the inhibitory compound was analyzed using disk diffusion tests. Results. All strains amplified the expected band for genus confirmation. Strains Bif 023 and Bif 013 were identified by DNA-sequencing as Bifidobacterium breve, with 97% homology. 17 strains were able to inhibit at least one of the pathogens tested. Escherichia coli ATCC 25922 was the most inhibited. It was determined that the strain Bif 023 is highly efficient as an antagonist strain due its ability to inhibit all the evaluated pathogens. Inhibition areas showed higher diameters than expected, suggesting an enhanced antagonist capacity of native strains. Bifidobacterium sp. was isolated from breast milk, meconium and newborn faeces which confirmed that this microorganism is human microbiota. Conclusion. Due to the high antagonist activity of most isolated bacteria, they could be playing an important protective function in the newborn, in particular strains Bif 013 and Bif 023, isolated from faeces. Other studies must be performed with these organisms to determine their probiotic potential as well as their use in the biocontrol industry due their activity against foodborne pathogens.