Colistin resistance in Escherichia coli and Klebsiella pneumoniae in humans and backyard animals in Ecuador

[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 preva- lence 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 con- tinual monitoring of the situation should be implemented.

[RESUMEN]. Objetivo. La colistina es un antibiótico de último recurso para tratar infecciones graves por bacterias gramneg- ativas. 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 reac- ción en cadena de la polimerasa. Resultados. Se obtuvo un total de 438 muestras fecales de 137 humanos, 147 cerdos y 154 pollos. La preva- lencia 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.

[RESUMO]. Objetivo. A colistina é um antibiótico de último recurso para o tratamento de infecções graves por bac- térias 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 isola- dos 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.

Synergy in penicillin, cephalosporin, amphenicols, and aminoglycoside against MDR S. aureus isolated from Camel milk

Abstract This study investigated the synergy testing of penicillin, cephalosporin, amphenicols, and aminoglycoside in the camel milk (n=768 samples), subsequently used for isolation of MDR S. aureus targeting mecA gene. Antibiotic susceptibility of S. aureus showed >90% isolates were sensitive to ciprofloxacin and trimethoprim and resistant against oxacillin, ampicillin, and cefoxitin. Further, 50-85% of the S. aureus were sensitive to gentamicin, oxytetracycline, and chloramphenicol and resistant against cefotaxime, vancomycin, and cefixime. Minimum inhibitory concentration (MIC) of cefotaxime, (C) and ampicillin (A) in combination with gentamicin (G) was reduced by 99.34% and 70.46%, respectively, while with chloramphenicol (Ch), reduction was 57.49% and 60%, respectively. In addition, the Fractional Inhibitory Concentration Index (FICI) of G+A, Ch+C and Ch+G combinations showed synergy against 80%, 60%, and 30% of MDR S. aureus, respectively. Similarly, C+A and Ch+G displayed indifferent interaction against 70 % and 30% of isolates, respectively, while the later showed additive interaction against 10% of MDR S. aureus. Altogether, our results described effective combination of gentamicin and chloramphenicol with ampicillin and cefotaxime to combat MDR S. aureus

The effect of the efflux pump inhibitor Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) on the susceptibility to imipenem and cefepime in clinical strains of Acinetobacter baumannii.

INTRODUCTION: In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. MATERIALS AND METHODS: A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). RESULTS: A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. CONCLUSION: In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii.

mcr-1-carrying Enterobacteriaceae isolated from companion animals in Brazil / Enterobacteriaceae portadora de mcr-1 em isolados clínicos de animais de companhia: primeiro relato no Brasil

Plasmid-mediated polymyxin resistance was first described in 2015, in China, in Escherichia coli carrying the mcr-1 (Mobile Colistin Resistance-1) gene. Since then, it has become a major public health challenge worldwide, representing a major threat to human and animal health. In addition, there are still few reports on the prevalence of mcr-1 in Enterobacteriaceae isolated from humans, animals and food. Therefore, the purpose of the study was to investigate the occurrence of the mcr-1 gene in bacterial isolates with phenotypic resistance to polymyxin B obtained from clinical specimens of companion animals. Phenotypic resistance to polymyxin B were determined by broth microdilution and the susceptibility profile to other antimicrobials (amikacin, amoxicillin/clavulanate, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, ertapenem, gentamicin, imipenem, marbofloxacin, meropenem, phosphomycin, piperacillin/tazobactam, tetracycline, ticarcillin/clavulanate, tobramycin and trimethoprim/sulfamethoxazole) by disc-diffusion agar method. The extraction of bacterial DNA was performed via heat shock followed by spectrophotometric evaluation. To verify the presence of mcr-1, the Polymerase Chain Reaction was employed using specific primers, followed by agarose gel electrophoresis. The positive isolates had the corresponding amplicons sequenced. In this study, there were identified the first isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. carrying the mcr-1 gene derived from specimens of companion animals in Brazil. Our results suggest the dissemination of resistance to polymyxins in the community and the environment, highlighting the need for surveillance and optimized treatment guidelines.(AU)

A resistência à polimixina mediada por plasmídeo teve sua primeira descrição em 2015, na China, em Escherichia coli portadora do gene mcr-1 (Mobile Colistin Resistance-1) e a partir de então tornou-se um grande desafio para a saúde pública em todo o mundo, constituindo uma grande ameaça à saúde humana e animal. Além disso, ainda existem poucos relatos sobre a prevalência de mcr-1 em Enterobacteriaceae isoladas de humanos, animais e alimentos. Sendo assim, o objetivo do estudo foi investigar a ocorrência do gene mcr-1 em isolados bacterianos com resistência fenotípica à polimixina B, oriundos de materiais clínicos de animais de companhia. A resistência fenotípica à polimixina B foi determinada por microdiluição em caldo e o perfil de sensibilidade aos demais antimicrobianos (amicacina, amoxicilina/clavulanato, ampicilina, ampicilina/sulbactam, aztreonam, cefazolina, cefepime, cefotaxima, cefoxitina, ceftazidima, ceftriaxona, cloranfenicol, ciprofloxacina, doxiciclina, ertapenem, gentamicina, imipinem, marbofloxacino, meropenem, fosfomicina, piperacilina/tazobactam, tetraciclina, ticarcilina/clavulanato, tobramicina sulfametoxazol/trimetoprim) foram determinados pelo método disco difusão. A extração do DNA bacteriano foi realizada via choque térmico, seguido de avaliação espectrofotométrica. Para a verificação da presença do mcr-1 foi utilizada a Reação em Cadeia da Polimerase com emprego de iniciadores específicos, seguida de eletroforese em gel de agarose. Os isolados positivos tiveram os correspondentes amplicons sequenciados. Nesse estudo foram identificados os primeiros isolados de Escherichia coli, Klebsiella spp. e Enterobacter spp. portadores do gene mcr-1 derivados de espécimes de animais de companhia no Brasil. Este estudo sugere a disseminação da resistência às polimixinas na comunidade e no meio ambiente, destacando a necessidade de vigilância e diretrizes otimizadas de tratamento.(AU)

mcr-1-carrying Enterobacteriaceae isolated from companion animals in Brazil / Enterobacteriaceae portadora de mcr-1 em isolados clínicos de animais de companhia: primeiro relato no Brasil

Plasmid-mediated polymyxin resistance was first described in 2015, in China, in Escherichia coli carrying the mcr-1 (Mobile Colistin Resistance-1) gene. Since then, it has become a major public health challenge worldwide, representing a major threat to human and animal health. In addition, there are still few reports on the prevalence of mcr-1 in Enterobacteriaceae isolated from humans, animals and food. Therefore, the purpose of the study was to investigate the occurrence of the mcr-1 gene in bacterial isolates with phenotypic resistance to polymyxin B obtained from clinical specimens of companion animals. Phenotypic resistance to polymyxin B were determined by broth microdilution and the susceptibility profile to other antimicrobials (amikacin, amoxicillin/clavulanate, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, ertapenem, gentamicin, imipenem, marbofloxacin, meropenem, phosphomycin, piperacillin/tazobactam, tetracycline, ticarcillin/clavulanate, tobramycin and trimethoprim/sulfamethoxazole) by disc-diffusion agar method. The extraction of bacterial DNA was performed via heat shock followed by spectrophotometric evaluation. To verify the presence of mcr-1, the Polymerase Chain Reaction was employed using specific primers, followed by agarose gel electrophoresis. The positive isolates had the corresponding amplicons sequenced. In this study, there were identified the first isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. carrying the mcr-1 gene derived from specimens of companion animals in Brazil. Our results suggest the dissemination of resistance to polymyxins in the community and the environment, highlighting the need for surveillance and optimized treatment guidelines.(AU)

A resistência à polimixina mediada por plasmídeo teve sua primeira descrição em 2015, na China, em Escherichia coli portadora do gene mcr-1 (Mobile Colistin Resistance-1) e a partir de então tornou-se um grande desafio para a saúde pública em todo o mundo, constituindo uma grande ameaça à saúde humana e animal. Além disso, ainda existem poucos relatos sobre a prevalência de mcr-1 em Enterobacteriaceae isoladas de humanos, animais e alimentos. Sendo assim, o objetivo do estudo foi investigar a ocorrência do gene mcr-1 em isolados bacterianos com resistência fenotípica à polimixina B, oriundos de materiais clínicos de animais de companhia. A resistência fenotípica à polimixina B foi determinada por microdiluição em caldo e o perfil de sensibilidade aos demais antimicrobianos (amicacina, amoxicilina/clavulanato, ampicilina, ampicilina/sulbactam, aztreonam, cefazolina, cefepime, cefotaxima, cefoxitina, ceftazidima, ceftriaxona, cloranfenicol, ciprofloxacina, doxiciclina, ertapenem, gentamicina, imipinem, marbofloxacino, meropenem, fosfomicina, piperacilina/tazobactam, tetraciclina, ticarcilina/clavulanato, tobramicina sulfametoxazol/trimetoprim) foram determinados pelo método disco difusão. A extração do DNA bacteriano foi realizada via choque térmico, seguido de avaliação espectrofotométrica. Para a verificação da presença do mcr-1 foi utilizada a Reação em Cadeia da Polimerase com emprego de iniciadores específicos, seguida de eletroforese em gel de agarose. Os isolados positivos tiveram os correspondentes amplicons sequenciados. Nesse estudo foram identificados os primeiros isolados de Escherichia coli, Klebsiella spp. e Enterobacter spp. portadores do gene mcr-1 derivados de espécimes de animais de companhia no Brasil. Este estudo sugere a disseminação da resistência às polimixinas na comunidade e no meio ambiente, destacando a necessidade de vigilância e diretrizes otimizadas de tratamento.(AU)

Quantitative analysis of the expression of fluconazole-resistant genes in strains of Candida albicans isolated from elderly people at their admission in an intensive care unit in Manizales, Colombia / [Análisis cuantitativo de la expresión de genes de resistencia a fluconazol en cepas de Candida albicans aisladas al ingreso de adultos mayores a una unidad de cuidados intensivos de Manizales, Colombia].

Introducción. Las infecciones oportunistas asociadas con Candida albicans han tenido gran repercusión en la salud pública por la mortalidad que generan en determinados grupos poblacionales. Aunque existen tratamientos farmacológicos disponibles, es evidente el aumento de la resistencia desarrollada por el agente patógeno, por lo que la determinación de los mecanismos de resistencia de las cepas presentes en las áreas hospitalarias es importante, ya que permitiría plantear mejores esquemas de tratamiento. Objetivo. Analizar la expresión de los genes ERG11, CDR1 y MDR1 en cepas de C. albicans aisladas de adultos mayores a su ingreso en la unidad de cuidados intensivos del Hospital Santa Sofía de Manizales, Colombia. Materiales y métodos. Se seleccionaron 29 muestras (21 resistentes y 8 sensibles) y se conformaron dos grupos de trabajo, uno de muestras con exposición al fluconazol y el otro sin esta. El ARN extraído se cuantificó mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real (RT-qPCR). Resultados. Se encontraron diferencias significativas en la expresión del gen MDR1 en el grupo de cepas de C. albicans resistentes. Dos de las cepas resistentes (104 y 62-2) expuestas al antifúngico presentaron valores muy elevados en la expresión de este gen. La expresión del ERG11 y del CDR1 no fue significativa en los grupos estudiados. Conclusión. El aumento de sobreexpresión del gen MDR1 indica que este puede ser el responsable de la resistencia; sin embargo, algunas cepas resistentes no sobreexpresaron los genes analizados, lo que indica que puede haber otros genes involucrados en la resistencia de las cepas estudiadas.

Introducción. Las infecciones oportunistas asociadas con Candida albicans han tenido gran repercusión en la salud pública por la mortalidad que generan en determinados grupos poblacionales. Aunque existen tratamientos farmacológicos disponibles, es evidente el aumento de la resistencia desarrollada por el agente patógeno, por lo que la determinación de los mecanismos de resistencia de las cepas presentes en las áreas hospitalarias es importante, ya que permitiría plantear mejores esquemas de tratamiento. Objetivo. Analizar la expresión de los genes ERG11, CDR1 y MDR1 en cepas de C. albicans aisladas de adultos mayores a su ingreso en la unidad de cuidados intensivos del Hospital Santa Sofía de Manizales, Colombia. Materiales y métodos. Se seleccionaron 29 muestras (21 resistentes y 8 sensibles) y se conformaron dos grupos de trabajo, uno de muestras con exposición al fluconazol y el otro sin esta. El ARN extraído se cuantificó mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real (RT-qPCR). Resultados. Se encontraron diferencias significativas en la expresión del gen MDR1 en el grupo de cepas de C. albicans resistentes. Dos de las cepas resistentes (104 y 62-2) expuestas al antifúngico presentaron valores muy elevados en la expresión de este gen. La expresión del ERG11 y del CDR1 no fue significativa en los grupos estudiados. Conclusión. El aumento de sobreexpresión del gen MDR1 indica que este puede ser el responsable de la resistencia; sin embargo, algunas cepas resistentes no sobreexpresaron los genes analizados, lo que indica que puede haber otros genes involucrados en la resistencia de las cepas estudiadas.

Detection of the mcr-1 gene in Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains isolated from broilers / Detecção do gene mcr-1 em estirpes de Escherichia coli Enteropatogênicas (EPEC) e Shigatoxigênicas (STEC) isoladas de frangos de corte

Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains are among the major pathotypes found in poultry and their products, which are capable of causing human enteric infections. Colistin has been claimed the drug of choice against diseases caused by multidrug-resistant Gram-negative bacteria (MDRGN) in humans. The mcr-1 gene was the first plasmidial gene that has been described to be responsible for colistin resistance and has also been detected in birds and poultry products. Our study aimed to detect the mcr-1 gene in enteropathogenic strains of E. coli in order to evaluate the resistance to colistin in broilers. The material was obtained from 240 cloacal samples and 60 broiler carcasses. The strains were isolated by the conventional bacteriological method and by the virulence genes, which characterize the enteropathogenic strains and resistance, and the samples were detected by polymerase chain reaction (PCR). Of the 213 isolated strains of E. coli, 57 (26.76%) were characterized as atypical EPEC and 35 (16.43%) as STEC. The mcr-1 gene was found in 3.5% (2/57) of the EPEC strains and 5.7% (2/35) of the STEC strains. In this study, it was possible to confirm that the mcr-1 resistance gene is already circulating in the broiler flocks studied and may be associated with the pathogenic strains.(AU)

Escherichia coli Enteropatogênica (EPEC) e Shigatoxigênica (STEC) estão entres os principais patotipos encontrados em aves e produtos avícolas que são capazes de causar doença entérica no homem. A colistina tem sido preconizada como droga de escolha para o tratamento de doenças causadas por bactérias Gram-negativas multirresistentes em humanos. O gene mcr-1 foi o primeiro gene plasmidial a ser descrito como responsável pela resistência a colistina e tem sido descrito em aves e produtos avícolas. Este estudo tem como objetivo a detecção do gene mcr-1 em estirpes de E. coli enteropatogênicas a fim de avaliar a resistência a colistina em frangos de corte. O material foi obtido a partir de 240 amostras cloacais e 60 carcaças de frango de corte. As estirpes foram isoladas pelo método bacteriológico convencional e os genes de virulência, que caracterizam as estirpes enteropatogênicas, e resistência foram detectados pela reação em cadeia pela polimerase (PCR). Das 213 estirpes de E. coli isoladas, 57 (26,76%) foram caracterizadas como EPEC atípica e 35 (16,43%) como STEC. O gene mcr-1 foi encontrado em 3,5% (2/57) das estirpes EPEC e 5,7% (2/35) das estirpes STEC. Neste estudo foi possível confirmar que o gene de resistência mcr-1 já está em circulação nos lotes de frango de corte estudados e pode estar associado às estirpes patogênicas.(AU)

Detection of the mcr-1 gene in Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains isolated from broilers / Detecção do gene mcr-1 em estirpes de Escherichia coli Enteropatogênicas (EPEC) e Shigatoxigênicas (STEC) isoladas de frangos de corte

Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains are among the major pathotypes found in poultry and their products, which are capable of causing human enteric infections. Colistin has been claimed the drug of choice against diseases caused by multidrug-resistant Gram-negative bacteria (MDRGN) in humans. The mcr-1 gene was the first plasmidial gene that has been described to be responsible for colistin resistance and has also been detected in birds and poultry products. Our study aimed to detect the mcr-1 gene in enteropathogenic strains of E. coli in order to evaluate the resistance to colistin in broilers. The material was obtained from 240 cloacal samples and 60 broiler carcasses. The strains were isolated by the conventional bacteriological method and by the virulence genes, which characterize the enteropathogenic strains and resistance, and the samples were detected by polymerase chain reaction (PCR). Of the 213 isolated strains of E. coli, 57 (26.76%) were characterized as atypical EPEC and 35 (16.43%) as STEC. The mcr-1 gene was found in 3.5% (2/57) of the EPEC strains and 5.7% (2/35) of the STEC strains. In this study, it was possible to confirm that the mcr-1 resistance gene is already circulating in the broiler flocks studied and may be associated with the pathogenic strains.(AU)

Escherichia coli Enteropatogênica (EPEC) e Shigatoxigênica (STEC) estão entres os principais patotipos encontrados em aves e produtos avícolas que são capazes de causar doença entérica no homem. A colistina tem sido preconizada como droga de escolha para o tratamento de doenças causadas por bactérias Gram-negativas multirresistentes em humanos. O gene mcr-1 foi o primeiro gene plasmidial a ser descrito como responsável pela resistência a colistina e tem sido descrito em aves e produtos avícolas. Este estudo tem como objetivo a detecção do gene mcr-1 em estirpes de E. coli enteropatogênicas a fim de avaliar a resistência a colistina em frangos de corte. O material foi obtido a partir de 240 amostras cloacais e 60 carcaças de frango de corte. As estirpes foram isoladas pelo método bacteriológico convencional e os genes de virulência, que caracterizam as estirpes enteropatogênicas, e resistência foram detectados pela reação em cadeia pela polimerase (PCR). Das 213 estirpes de E. coli isoladas, 57 (26,76%) foram caracterizadas como EPEC atípica e 35 (16,43%) como STEC. O gene mcr-1 foi encontrado em 3,5% (2/57) das estirpes EPEC e 5,7% (2/35) das estirpes STEC. Neste estudo foi possível confirmar que o gene de resistência mcr-1 já está em circulação nos lotes de frango de corte estudados e pode estar associado às estirpes patogênicas.(AU)

Diverse Commensal Escherichia coli Clones and Plasmids Disseminate Antimicrobial Resistance Genes in Domestic Animals and Children in a Semirural Community in Ecuador.

The increased prevalence of antimicrobial resistance (AMR) among Enterobacteriaceae has had major clinical and economic impacts on human medicine. Many of the multidrug-resistant (multiresistant) Enterobacteriaceae found in humans are community acquired, and some of them are possibly linked to food animals (i.e., livestock raised for meat and dairy products). In this study, we examined whether numerically dominant commensal Escherichia coli strains from humans (n = 63 isolates) and domestic animals (n = 174 isolates) in the same community and with matching phenotypic AMR patterns were clonally related or shared the same plasmids. We identified 25 multiresistant isolates (i.e., isolates resistant to more than one antimicrobial) that shared identical phenotypic resistance patterns. We then investigated the diversity of E. coli clones, AMR genes, and plasmids carrying the AMR genes using conjugation, replicon typing, and whole-genome sequencing. All of the multiresistant E. coli isolates (from children and domestic animals) analyzed had at least 90 or more whole-genome SNP differences between one another, suggesting that none of the strains was recently transferred. While the majority of isolates shared the same antimicrobial resistance genes and replicons, DNA sequencing indicated that these genes and replicons were found on different plasmid structures. We did not find evidence of the clonal spread of AMR in this community: instead, AMR genes were carried on diverse clones and plasmids. This presents a significant challenge for understanding the movement of AMR in a community.IMPORTANCE Even though Escherichia coli strains may share nearly identical phenotypic AMR profiles and AMR genes and overlap in space and time, the diversity of clones and plasmids challenges research that aims to identify sources of AMR. Horizontal gene transfer appears to play a more significant role than clonal expansion in the spread of AMR in this community.

Lipid core nanoparticles as a broad strategy to reverse fluconazole resistance in multiple Candida species.

Fungal resistance is the major problem related to fluconazole treatments. This study aims to develop innovative lipid core nanocapsules and nanostructured lipid carriers containing fluconazole, to study in vitro antifungal activity and to assess the possibility of resistance reversion in Candida albicans, C. glabrata, C. krusei, and C. tropicalis isolates. The action mechanism of nanoparticles was investigated through efflux pumps and scanning electron microscopy studies. The lipid core nanocapsules and nanostructured lipid carriers were prepared by interfacial deposition of preformed polymer and high-pressure homogenization methods, respectively. Both nanostructures presented sizes below 250 nm, SPAN < 1.6, negative zeta potential, pH slightly acid, high drug content and controlled drug release. The nanostructured lipid carriers were unable to reverse the fungal resistance. Lipid core nanoparticles displayed advantages such as a reduction in the effective dose of fluconazole and resistance reversion in all isolates tested - with multiple mechanisms of resistance. The main role of the supramolecular structure and the composition of the nanoparticles on antifungal mechanisms of action were discussed. The results achieved through this study have an impact on clinical therapy, with a potential application in the treatment of fungal infections caused by resistant isolates of Candida spp.

Gene regulatory network inference and analysis of multidrug-resistant Pseudomonas aeruginosa

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

Multidrug resistant and ESBL-producing Salmonella spp. isolated from poultry / Salmonella spp. produtora de ESBL e multirresistente a drogas isolada de frangos

Salmonella spp. is one of the main agents responsible for foodborne infection in humans, and products of poultry origin are the most common infection sources. Studies have shown the occurrence of antimicrobials resistant Salmonella spp. in animal products. The Extended Spectrum β-Lactamase (ESBL) are enzymes that confer to bacteria the ability to hydrolyze cephalosporin with an oximino side chain and monobactams. This study aimed to investigate antimicrobial resistance profile, identify phenotypes and genotypes for multiple drug resistance (MDR) and that produce ESBL from isolates of Salmonella spp. in the broiler production chain. We used samples of Salmonella spp. (n=11) isolates from poultry, poultry products and poultry-source environment from the state of Maranhão - Brazil. The isolates of Salmonella spp. assessed showed genotypical and phenotypical characteristics of MDR. The results show that 72.72% (08/11) of the strains presented the phenotypic profile for ESBL production. The isolates showed positivity to at least 13.64% (03/22) of the genes studied and the highest frequencies were observed in genes sul1 (73%), dfrA12 (55%), blaCTX-M (55%), tetA, tetB and tetC, with 45%. In conclusion, the strains of Salmonella spp. isolates present genotypic and phenotypic characteristics for MDR and ESBL production, demonstrating the dissemination risk of these microorganisms through the food chain.

Salmonella spp. é um dos principais agentes responsáveis por infecção de origem alimentar em humanos, sendo os produtos de origem avícola apontados como as fontes de infecção mais frequentes. Estudos demonstraram a ocorrência de Salmonella spp. resistente a antimicrobianos em produtos de origem animal. As β-lactamases de espectro extendido (ESBL) são enzimas que conferem às bactérias a capacidade de hidrolisar cefalosporinas com uma cadeia lateral oximino e monobactâmicos. Este estudo objetivou investigar o perfil de resistência a antimicrobianos, identificar fenótipos e genótipos de multirresistência a drogas (MDR), e produção de ESBL em isolados de Salmonella spp. provenientes da cadeia produtiva de frangos de corte. Foram utilizadas amostras de Salmonella spp. (n=11) isoladas de aves, produtos e ambiente de origem avícola do estado do Maranhão - Brasil. Os isolados de Salmonella spp. avaliados apresentaram resistência múltipla a drogas tanto genotípica quanto fenotipicamente. Os resultados mostram que 72,72% (08/11) das cepas apresentaram perfil fenotípico de produção de ESBL. Os isolados apresentaram positividade a pelo menos 13,64% (03/22) dos genes pesquisados, sendo as maiores frequências observadas nos genes sul1 (73%), dfrA12 (55%), blaCTX-M (55%), tetA, tetB e tetC, com 45%. Conclui-se que as cepas de Salmonella spp. isoladas apresentam características genotípicas e fenotípicas de multirresistência a drogas e produção de ESBL, demonstrando o risco de disseminação destes microrganismos ao longo da cadeia alimentar.

Multidrug resistant and ESBL-producing Salmonella spp. isolated from poultry / Salmonella spp. produtora de ESBL e multirresistente a drogas isolada de frangos

Salmonella spp. is one of the main agents responsible for foodborne infection in humans, and products of poultry origin are the most common infection sources. Studies have shown the occurrence of antimicrobials resistant Salmonella spp. in animal products. The Extended Spectrum β-Lactamase (ESBL) are enzymes that confer to bacteria the ability to hydrolyze cephalosporin with an oximino side chain and monobactams. This study aimed to investigate antimicrobial resistance profile, identify phenotypes and genotypes for multiple drug resistance (MDR) and that produce ESBL from isolates of Salmonella spp. in the broiler production chain. We used samples of Salmonella spp. (n=11) isolates from poultry, poultry products and poultry-source environment from the state of Maranhão - Brazil. The isolates of Salmonella spp. assessed showed genotypical and phenotypical characteristics of MDR. The results show that 72.72% (08/11) of the strains presented the phenotypic profile for ESBL production. The isolates showed positivity to at least 13.64% (03/22) of the genes studied and the highest frequencies were observed in genes sul1 (73%), dfrA12 (55%), blaCTX-M (55%), tetA, tetB and tetC, with 45%. In conclusion, the strains of Salmonella spp. isolates present genotypic and phenotypic characteristics for MDR and ESBL production, demonstrating the dissemination risk of these microorganisms through the food chain.(AU)

Salmonella spp. é um dos principais agentes responsáveis por infecção de origem alimentar em humanos, sendo os produtos de origem avícola apontados como as fontes de infecção mais frequentes. Estudos demonstraram a ocorrência de Salmonella spp. resistente a antimicrobianos em produtos de origem animal. As β-lactamases de espectro extendido (ESBL) são enzimas que conferem às bactérias a capacidade de hidrolisar cefalosporinas com uma cadeia lateral oximino e monobactâmicos. Este estudo objetivou investigar o perfil de resistência a antimicrobianos, identificar fenótipos e genótipos de multirresistência a drogas (MDR), e produção de ESBL em isolados de Salmonella spp. provenientes da cadeia produtiva de frangos de corte. Foram utilizadas amostras de Salmonella spp. (n=11) isoladas de aves, produtos e ambiente de origem avícola do estado do Maranhão - Brasil. Os isolados de Salmonella spp. avaliados apresentaram resistência múltipla a drogas tanto genotípica quanto fenotipicamente. Os resultados mostram que 72,72% (08/11) das cepas apresentaram perfil fenotípico de produção de ESBL. Os isolados apresentaram positividade a pelo menos 13,64% (03/22) dos genes pesquisados, sendo as maiores frequências observadas nos genes sul1 (73%), dfrA12 (55%), blaCTX-M (55%), tetA, tetB e tetC, com 45%. Conclui-se que as cepas de Salmonella spp. isoladas apresentam características genotípicas e fenotípicas de multirresistência a drogas e produção de ESBL, demonstrando o risco de disseminação destes microrganismos ao longo da cadeia alimentar.(AU)

Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna

Escalating burden of antibiotic resistance that has reached new heights present a grave concern to mankind. As the problem is no longer confined to clinics, we hereby report identification of a pandrug resistant Escherichia coli isolate from heavily polluted Delhi stretch of river Yamuna, India. E. coli MRC11 was found sensitive only to tobramycin against 21 antibiotics tested, with minimum inhibitory concentration values >256 µg/mL for amoxicillin, carbenicillin, aztreonam, ceftazidime and cefotaxime. Addition of certain heavy metals at higher concentrations were ineffective in increasing susceptibility of E. coli MRC11 to antibiotics. Withstanding sub-optimal concentration of cefotaxime (10 µg/mL) and mercuric chloride (2 µg/mL), and also resistance to their combinatorial use, indicates better adaptability in heavily polluted environment through clustering and expression of resistance genes. Interestingly, E. coli MRC11 harbours two different variants of blaTEM (blaTEM-116 and blaTEM-1 with and without extended-spectrum activity, respectively), in addition to mer operon (merB, merP and merT) genes. Studies employing conjugation, confirmed localization of blaTEM-116, merP and merT genes on the conjugative plasmid. Understanding potentialities of such isolates will help in determining risk factors attributing pandrug resistance and strengthening strategic development of new and effective antimicrobial agents.(AU)

Primary antibiotic resistance and its relationship with cagA and vacA genes in Helicobacter pylori isolates from Algerian patients

The epidemiology of Helicobacter pylori resistance to antibiotics is poorly documented in Africa and especially in Algeria. The aim of our study was to determine the antibiotic resistance rates, as well as its possible relationship with VacA and CagA virulence markers of isolates from Algerian patients. One hundred and fifty one H. pylori isolate were obtained between 2012 and 2015 from 200 patients with upper abdominal pain. Antimicrobial susceptibility testing was performed for amoxicillin, clarithromycin, metronidazole, ciprofloxacin, rifampicin and tetracycline. Molecular identification of H. pylori and the detection of vacA and cagA genes were performed using specific primers. We found that H. pylori was present in 83.5% of collected biopsies, 54.9% of the samples were cagA positive, 49.67% were vacA s1m1, 18.30% were vacA s1m2 and 25.49% were vacA s2m2. Isolates were characterized by no resistance to amoxicillin (0%), tetracycline (0%), rifampicin (0%), a high rate of resistance to metronidazole (61.1%) and a lower rate of resistance to clarithromycin (22.8%) and ciprofloxacin (16.8%). No statically significant relationship was found between vagA and cagA genotypes and antibiotic resistance results (p > 0.5) except for the metronidazole, which had relation with the presence of cagA genotype (p = 0.001).(AU)

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Meropenem Efflux in Pseudomonas aeruginosa at a Tertiary Care Hospital in Jamaica / Eflujo de meropenem en Pseudomonas aeruginosa en un hospital de atención terciaria en Jamaica

ABSTRACT Objective: Several mechanisms account for carbapenem resistance in Pseudomonas (P) aeruginosa which is an emerging problem at a tertiary care hospital (TCH) in Jamaica. The observed pattern of carbapenem resistance that results from efflux mechanisms is unique because it is specific to meropenem (MEM). An investigation of efflux as a mechanism of carbapenem resistance was needed as the information obtained could inform therapeutic and infection control strategies. Methods: At the Microbiology Laboratory of a TCH in Jamaica, from May 2009 to March 2011, of 105 multidrug-resistant Gram-negative bacilli isolated from clinical specimens submitted for routine identification and susceptibility testing, all the MEM-resistant P aeruginosa isolates (a total of 10) were selected. They were tested for efflux using the efflux inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in a method described by Giske et al in 2008. Results: This study detected evidence of MEM efflux in 80% of MEM-resistant P aeruginosa implicated in nosocomial infections at this TCH in Jamaica, using the PAβN inhibition assay. Meropenem-efflux-positive isolates belonged to two unrelated chromosomal lineages. Conclusion: These results underscored the need for improved surveillance and control to prevent this mechanism from emerging in further P aeruginosa strains.

RESUMEN Objetivo: Varios mecanismos explican la resistencia al carbapenem en Pseudomonas (P) Aeruginosa - un problema que recientemente se está presentando en un hospital de atención terciaria (HAT) en Jamaica. El patrón observado de resistencia al carbapenem que resulta de los mecanismos de eflujo es único, porque es específico del meropenem (MEM). Fue necesario realizar una investigación del eflujo como mecanismo de resistencia al carbapenem, ya que la información obtenida podría usarse para las estrategias de terapia y de control de la infección. Métodos: En el Laboratorio de Microbiología de un HAT en Jamaica, de mayo de 2009 a marzo de 2011, de 105 bacilos gram-negativos polifármacorresistentes aislados de especímenes clínicos presentados para identificación de rutina y pruebas de susceptibilidad, se seleccionaron todos los aislados de P aeruginosa (un total de 10) resistentes a MEM. Estos aislados fueron sometidos a prueba de detección de eflujo, usando el inhibidor de eflujo de fenilalanina-arginina-β-naftilamida (PAβN) en un método descrito por Giske et al en 2008. Resultados: Este estudio detectó evidencia de eflujo de MEM en el 80% de P aeruginosa resistente a MEM implicado en infecciones nosocomiales en este HAT de Jamaica, usando el ensayo de inhibición PAβN. Los aislados positivos al eflujo de meropenem pertenecían a dos linajes cromosómicos no relacionados. Conclusión: Estos resultados subrayaron la necesidad de mejorar la vigilancia y el control para prevenir que este mecanismo vuelva a producirse en nuevas cepas de P aeruginosa.

Resistencia plasmídica a colistin por el gen mcr-1 en Enterobacteriaceae en Paraguay / Plasmid-Mediated Colistin Resistance Gene mcr-1 in Enterobacteriaceae in Paraguay

La resistencia a las polimixinas mediada por plásmidos (gen mcr-1) representa una amenaza para la salud pública, puesto que colistina es utilizada en la práctica médica como una de las últimas alternativas para el tratamiento de gérmenes multiresistentes. Este estudio describe la circulaciónde cepas de Enterobacterias que portan este gen de resistencia, aisladas de pacientes hospitalizados, así como también de la comunidad. Los hallazgos de la Red de Vigilancia de la Resistencia a los Antimicrobianos-Paraguay fueron de casi el 5 % (4,7) en cepas remitidas con criterio de sospecha, siendo las especies involucradas Escherichiacoli, Klebsiella pneumoniae y Salmonella Schwarzengrund. Además, por métodos moleculares se confirmaron en todas ellas la portación de otros genes de resistencia (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) asociados al mcr-1. Palabras claves: Enterobacterias, resistencia, colistina, mcr-1.

Resistance to polymyxins mediated by plasmids (mcr-1 gene) represents a threat to public health, since colistin is used in medical practice, as one of the last alternatives, for the treatment of multi-resistant germs. This study describes the circulation of strains of Enterobacteria that carry this resistance gene, isolated from hospitalized patients, as well as from the community. The findings of the Red de Vigilancia de la Resistencia a los Antimicrobianos­Paraguay were almost 5% (4.7) in strains submitted with suspicion criteria; the species involved being Escherichia coli, Klebsiella pneumoniae and Salmonella Schwarzengrund. In addition, molecular methods confirmed in all of them the carrying of other resistance genes (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) associated with mcr-1. Key words: Enterobacteria, resistance, colistin, mcr-1.

An epidemiological study of drug resistance and resistance genes in bovine Escherichia coli isolates in Heilongjiang Province of China

Background: To explore the epidemiology of bovine multidrug-resistant Escherichia coli isolates and resistance genes in Heilongjiang province of China. This study examined the prevalence of genes in bovine E. coli isolates, which confer resistance to antibiotics that are commonly used in the clinic, in regions of Baiquan, Shangzhi, and Songbei of Harbin. The purpose of the study was to investigate the epidemiology of the main resistance genes of bovine E. coli isolates in clinical veterinary medicine, and to provide a theoretical basis for preventing the spread of drug-resistant bacteria, as well as for rational drug use.Materials, Methods & Results: The sensitivity of 105 isolates to 22 antibiotics was determined using the KirbyBauer disk diffusion method, and the distribution of 19 kinds of common drug resistance genes was investigated using Polymerase Chain Reaction. The results showed that the resistance rate to nine antibiotics was over 50%, including rifampin (84.76%), ampicillin (73.58%), tetracycline (69.52%), and sulfisoxazole (59.05%). In total, 105 strains of bovine E. coli presented 21 spectra of drug resistance, including eight strains (7.62%, 8/105) that were resistant to one antibiotic and four strains (3.81%, 4/105) that were resistant to 21 antibiotics. The resistance gene detection results showed that the streptomycin-resistance gene strA was found in 73 isolates, accounting for 69.52% of the isolates, followed by the sulfanilamide-resistance genes sul3/sul2 and the aminoglycoside-resistance gene aphA, which accounted for 57.14%, 51.43%, and 50.48%, respectively, of the isolates.[...]