Discovery, structure and function of plasmid mediated shufflon / 生物工程学报

Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.

A bioinformatics approach revealed the transcription factors of Helicobacter pylori pathogenic genes and their regulatory network nodes

BACKGROUND: Helicobacter pylori is a chronic pathogenic bacteria that causes gastric mucosal damage through various host-related and pathogen-related factors. Thus, a single gene research cannot fully explain its pathogenicity. PURPOSE OF STUDY: It is necessary to establish a Helicobacter pylori pathogenic gene transcription factor regulatory network (TFRN) and study its central nodes. RESULTS: The expression data of Helicobacter pylori pathogenic genes were obtained through GEO Datasets of NCBI. The genes were screened using linear model-empirical Bayesian statistics in R language Limma package combined with the conventional t-test; the results identified 1231 differentially expressed genes. The functional analysis (gene ontology-analysis) and signal pathway analysis (pathway-analysis) of differentially expressed genes were performed using the DAVID and KEGG databases, respectively. The pathogenic gene regulatory network was constructed by integrating transcriptional regulatory element database (TRED); the disease-related analysis of the pathogenic genes was conducted using the DAVID annotation tool. Five pathogenic genes (Nos2, Il5, Colla1, Tnf, and Nfkb1) and their transcription factors (Jun, Cebpa, Egrl, Ppara, and Il6) were found to suppress the host immune function and enhance the pathogenicity of Helicobacter pylori by regulating the host immune system. CONCLUSIONS: This effect was largely mediated via three signaling pathways: Tnf pathway, PI3K Akt pathway, and Jak­STAT pathway. The pathogenicity of Helicobacter pylori is closely related to the body's immune and inflammatory system. A better understanding of the correlation of the pathogenic factors with the host immune and inflammatory factors may help to determine the precise pathogenic mechanism of H. pylori infection.

Coagulase-negative staphylococci: a 20-year study on the antimicrobial resistance profile of blood culture isolates from a teaching hospital

ABSTRACT The increasing rates of nosocomial infection associated with coagulase-negative staphylococci (CoNS) were the rationale for this study, aiming to categorize oxacillin-resistant CoNS species recovered from blood culture specimens of inpatients at the UNESP Hospital das Clínicas in Botucatu, Brazil, over a 20-year period, and determine their sensitivity to other antimicrobial agents. The mecA gene was detected in 222 (74%) CoNS samples, and the four types of staphylococcal chromosomal cassette mec (SCCmec) were characterized in 19.4%, 3.6%, 54.5%, and 14.4% of specimens, respectively, for types I, II, III, and IV. Minimal inhibitory concentration (MIC) values to inhibit 50% (MIC50) and 90% (MIC90) of specimens were, respectively, 2 and >256 µL/mL for oxacillin, 1.5 and 2 µL/mL for vancomycin, 0.25 and 0.5 µL/mL for linezolid, 0.094 and 0.19 µL/mL for daptomycin, 0.19 and 0.5 µL/mL for quinupristin/dalfopristin, and 0.125 and 0.38 µL/mL for tigecycline. Resistance to oxacillin and tigecycline and intermediate resistance to quinupristin/dalfopristin were observed. Eight (2.7%) of all 300 CoNS specimens studied showed reduced susceptibility to vancomycin. Results from this study show high resistance rates of CoNS to antimicrobial agents, reflecting the necessity of using these drugs judiciously and controlling nosocomial dissemination of these pathogens.

Prevalence of Opportunistic Pathogens and Diversity of Microbial Communities in the Water System of a Pulmonary Hospital / 生物医学与环境科学（英文）

Objective@#Our objective was to investigate the occurrence of opportunistic pathogens and characterize the bacterial community structures in the water system of a pulmonary hospital.@*Methods@#The water samples were collected from automatic and manual faucets in the consulting room, treatment room, dressing room, respiratory ward, and other non-medical rooms in three buildings of the hospital. Quantitative polymerase chain reaction was used to quantify the load of several waterborne opportunistic pathogens and related microorganisms, including spp., spp., and . Illumina sequencing targeting 16S rRNA genes was performed to profile bacterial communities.@*Results@#The occurrence rates of spp., spp., and were 100%, 100%, and 76%, respectively in all samples. Higher occurrence rates of were observed in the outpatient service building (building 1, 91.7%) and respiration department and wards (building 2, 80%) than in the office building (building 3), where no was found. were more abundant in automatic faucets (average 2.21 × 10 gene copies/L) than in manual faucets (average 1.03 × 10 gene copies/mL) ( < 0.01). , , , , , and were the dominant bacterial phyla. Disinfectant residuals, nitrate, and temperature were found to be the key environmental factors driving microbial community structure shifts in water systems.@*Conclusion@#This study revealed a high level of colonization of water faucets by opportunistic pathogens and provided insight into the characteristics of microbial communities in a hospital water system and approaches to reduce risks of microbial contamination.

Virulence genes in Escherichia coli isolates from commercialized saltwater mussels Mytella guyanensis (Lamarck, 1819) / Presença de genes de virulência em isolados de Escherichia coli provenientes de sururu Mytella guyanensis (Lamarck, 1819) comercializado

Abstract The isolation of Escherichia coli from food is a major concern. Pathogenic strains of these bacteria cause diseases which range from diarrhea to hemolytic-uremic syndrome. Therefore the virulence genes in E. coli isolates from the mussel ( Mytella guyanensis) commercialized in Cachoeira, Bahia, Brazil were investigated. Samples were purchased from four vendors: two from supermarkets and two from fair outlets. They were conditioned into isothermal boxes with reusable ice and transported to the laboratory for analysis. E. coli strains were isolated in eosin methylene blue agar, preserved in brain-heart infusion medium with 15% glycerol and stored at -20 °C, after microbiological analysis. Virulence genes in the isolated strains were identified by specific primers, with Polymerase Chain Reaction. Twenty-four isolates were obtained, with a prevalence of elt gene, typical from enterotoxigenic infection, in 75% of the isolates. The stx and bfpA genes, prevalent in enterohemorragic and enteropathogenic E. coli, respectively, were not detected. The occurrence of elt virulence-related gene in the E. coli isolates of Mytella guyanensis reveals urgent improvement in food processing, including good handling practices, adequate storage and cooking before consumption, to ensure consumer's health.

Resumo O isolamento de Escherichia coli a partir de alimentos é uma grande preocupação, pois cepas patogênicas desta bactéria podem causar desde diarreia até síndrome hemolítico-urêmica. Diante do exposto, o objetivo do trabalho foi pesquisar genes de virulência em isolados de Escherichia coli provenientes do sururu Mytella guyanensis comercializado na cidade de Cachoeira, Bahia, Brasil. As amostras foram adquiridas de quatro comerciantes, sendo duas de mercados e duas em pontos de venda na feira livre da cidade de Cachoeira, acondicionadas em caixas isotérmicas com gelo reutilizável e transportadas até o laboratório para a análise. Após a análise microbiológica, as cepas de Escherichia coli foram isoladas em ágar Eosina Azul de Metileno e preservadas em caldo Brian Heart Infusion e glicerol a 15% e mantidas a - 20° C. A identificação dos genes de virulência nas cepas isoladas foi realizada utilizando primers específicos, por meio da Reação em Cadeia da Polimerase. Foram obtidos 24 isolados de Escherichia coli, destes a prevalência do gene elt , característico de Escherichia coli enterotoxigênica, foi de 75% dos isolados. Não houve a detecção dos genes stx e bfpA nos isolados, os quais são prevalentes nas cepas de Escherichia coli enterohemorrágica e Escherichia coli enteropatogênica, respectivamente. A presença do gene elt relacionado à virulência de Escherichia coli nos isolados de Mytella guyanensis revela a necessidade da melhoria no processamento, incluindo boas práticas de manipulação, armazenamento adequado e cocção previa ao consumo, visando a garantia da saúde do consumidor.

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

Resistencia a la meticilina y producción de biopelícula en aislamientos clínicos de Staphylococcus aureus y Staphylococcus coagulasa negativa en México / Methicillin resistance and biofilm production in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus in México

Resumen Introducción. Las infecciones por Staphylococcus aureus y Staphylococcus coagulasa negativa multirresistentes a los antibióticos y asociadas con la atención en salud tienen un gran impacto epidemiológico por su alta morbimortalidad; además, se han relacionado con la formación de biopelículas, lo cual también se asocia con la resistencia a los antimicrobianos. Objetivo. Determinar la resistencia a la meticilina y cuantificar la producción de biopelículas para establecer su posible relación con los aislamientos clínicos de S. aureus y Staphylococcus coagulasa negativa. Materiales y métodos. Se estudiaron 11 cepas de S. aureus y 12 de Staphylococcus coagulasa negativa. La resistencia a la meticilina se determinó con discos de cefoxitina tomando como valores de referencia los estándares del Clinical Laboratory Standards Institute (CLSI) de 2018. La producción de biopelícula se cuantificó con cristal violeta. Los genes mecA e icaADBC se identificaron mediante reacción en cadena de la polimerasa (PCR), y se hizo un análisis bivariado con la prueba de ji al cuadrado y el coeficiente V de Cramér, utilizando el programa SPSS™, versión 20.0. Resultados. Nueve cepas de S. aureus fueron resistentes a la meticilina (SARM) y dos fueron sensibles. Ocho cepas de Staphylococcus coagulasa negativa fueron resistentes y cuatro fueron sensibles. El genotipo mecA se encontró en ocho de las nueve cepas de S. aureus y en seis de las ocho de Staphylococcus coagulasa negativa resistentes a meticilina. Todas las cepas formaron biopelícula. Diez cepas de S. aureus y 11 de Staphylococcus coagulasa negativa presentaron el genotipo icaADCB. No se encontró asociación entre la resistencia a meticilina y la formación de biopelícula. Conclusiones. La cefoxitina es suficiente para determinar el fenotipo resistente a meticilina y se asoció con el genotipo mecA. Las cepas resistentes a la meticilina y poseedoras del gen mecA pueden presentar un mecanismo de resistencia alterno. Los dos grupos de cepas formadoras de biopelícula se relacionaron con la presencia del operón icaADCB. La formación de biopelícula y la resistencia a la meticilina se expresaron como características independientes en los dos grupos de cepas.

Abstract Introduction: Infections associated with health care caused by S. aureus and coagulase- negative Staphylococci multi-resistant to antibiotics cause a high epidemiological impact due to their high morbidity and mortality. Biofilm formation, which has been associated with antimicrobial resistance, can also occur. Objectives: To determine methicillin resistance and to quantify the biofilm production to establish if there is a relationship in clinical isolates of S. aureus and coagulase-negative Staphylococci. Material and methods: A total of 11 strains of S. aureus and 12 of coagulase-negative Staphylococci were studied. Methicillin resistance was determined with cefoxitin discs and the Clinical Laboratory Standards Institute (CSLI), 2018 reference values. Biofilm production was quantified by the crystal violet method. The mecA and icaADBC genes were identified by PCR. A bivariate analysis was performed with chi-square (c2) and Cramér's V statistical tests, using SPSS™, version 20.0 software. Results: Nine S. aureus strains were methicillin-resistant and two were sensitive. Eight coagulase-negative Staphylococci strains were resistant and four were sensitive. The mecA genotype was found in eight of the nine S. aureus resistant strains and six of eight resistant coagulase-negative Staphylococci. All strains formed biofilms. Ten strains of S. aureus and 11 of coagulase-negative Staphylococci presented the icaADCB genotype. No association was found between methicillin-resistance and biofilm formation. Conclusions: Cefoxitin is enough to define the resistance phenotype and is associated with the mecA genotype. All strains formed biofilms and were related to the presence of the icaADCB operon. Biofilm formation and methicillin resistance were independent features in both groups of strains.

Diversity and abundance of denitrifiers during cow manure composting / Diversidad y abundancia de desnitrificadores durante el compostaje del estiércol de vaca

Diversity and abundance of the denitrifying genes nirK, nirS and nosZ were investigated in cow manure compost using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time quantitative PCR (qPCR), respectively. These three genes were detected in all the stages of the composting process. Phylogenetic analysis showed that the nirK gene was closely related to Rhizobiales, Burkholderiales, the nirS gene was closely related to Pseudomonadales and Burkholderiales, and the nosZ gene was closely related to Rhodospirillales, Rhizobiales, Pseudomonadales, and Alteromonadales. qPCR results showed that the abundance of these three genes (nirK, nirS and nosZ) reached the peak value in the late thermophilic stage of composting and abundance of the nirK gene was higher than that of the nosZ gene and the nirS gene. Redundancy analysis (RDA) showed that the diversity of the nirK and nirS genes was significantly correlated with ammonium (p < 0.05), the diversity of the nosZ gene was significantly correlated with pH (p < 0.05) and the abundance of the nirK nirS and nosZ genes was significantly correlated with temperature (p< 0.05).

La diversidad y la abundancia de los genes desnitrificadores nirK, nirS, nosZ en el compost de estiércol de vaca se investigaron por medio de la reacción en cadena de la polimerasa seguida de electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) y por PCR cuantitativa (qPCR) en tiempo real, respectivamente. Estos 3 genes fueron detectados durante todas las fases del compostaje. El análisis filogenético mostró estrecha relación del gen nirK con Rhizobiales y Burkholderiales, del gen nirS con Pseudomonadales y Burkholderiales y del gen nosZ con Rhodospirillales, Rhizobiales, Pseudomonadales y Alteromonadales. Los resultados de la qPCR mostraron que la abundancia de los genes nirK, nirSy nosZ alcanzó el valor máximo en la fase termofílica tardía del compostaje, y que la abundancia del gen nirK era más elevada que los de los genes nosZ y nirS. El análisis de redundancia (RDA) mostró que la diversidad de los genes nirK y nirS estaba significativamente correlacionada con la concentración de amonio (p<0,05), mientras que la del gen nosZ estaba significativamente correlacionada con el pH (p<0,05). También mostró que la abundancia de los genes nirK, nirS y nosZ estaba significativamente correlacionada con la temperatura (p<0,05).

Identification of Brucella abortus biovar 4 of bovine origin in Colombia / Identificación de Brucella abortus biovar 4 de origen bovino en Colombia

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest -Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

El objetivo de este estudio fue identificar 12 aislamientos de Brucella abortus de origen bovino procedentes del departamento de Narino, Colombia, hasta la descripción de biovar. Estos aislamientos conforman la colección del Banco de Germoplasma de Microorganismos de Interés en Salud Animal, Bacterias y Virus. La identificación se hizo mediante métodos convencionales, como la descripción morfológica macro y microscópica de actividad enzimática, de perfiles bioquímicos, de utilización de sustratos y de sensibilidad a colorantes. Se hizo una caracterización genotipica complementaria mediante PCR múltiple para Brucella abortus, Brucella melitensis, Brucella ovisy Brucella suis-eritritol (AMOS-ERY-PCR); RFLP-/S7II; hibridación Southern blot y análisis multi-locus de repeticiones en tándem de número variable (MLVA), empleando como marcadores moleculares el gen ery, la secuencia de inserción /S711 y el número variable de repeticiones en tándem (VNTR). Los resultados de la caracterización fenotípica y molecular permitieron identificar 12 aislamientos de campo como B. abortus biovar 4 y diferenciar cepas de campo de cepas vacunales. Este es el primer estudio de identificación fenotípica y molecular de aislamientos de B. abortus en Colombia. Por su importancia taxonómica y epidemiológica, la identificación de estos aislamientos hasta el nivel de biovar permitirá disponer de recursos genéticos que se pueden emplear como cepas de referencia en futuras investigaciones. Estos resultados pueden considerarse como una base para la identificación de biotipos no reportados en el país y podrán ser utilizados en programas de monitoreo y vigilancia de la brucelosis bovina en Colombia.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province / Diversidad genética de Mycoplasma hyopneumoniae en la provincia de Mendoza

En Argentina, la neumonía enzoótica porcina (NEP) es altamente prevalente y se han identificado diferentes tipos genéticos de Mycoplasma hyopneumoniae. Sin embargo, se carece de información acerca de la prevalencia de NEP y de otros aspectos epidemiológicos de esta entidad en la provincia de Mendoza. En esta investigación se usó un análisis multilocus de regiones repetidas en tándem (MLVA) de los loci P97 R1, P97 R1A y P146 R3 para evaluar la diversidad genética de M. hyopneumoniae a partir de muestras clínicas de cerdos de cinco granjas localizadas en diferentes distritos de la provincia de Mendoza. M. hyopneumoniae pudo ser tipificado a partir de 27 muestras de lavado broncoalveolar (LBA) y se identificaron 8 diferentes MLVA-tipos. Este es el primer informe acerca de la diversidad genética de M. hyopneumoniae en Mendoza. Los resultados obtenidos permiten describir de manera más acabada la diversidad genética de este agente en nuestro país.

Mutaciones del gen ARN ribosómico 23S de Helicobacter pylori asociadas con resistencia a claritromicina en pacientes atendidos en una unidad de endoscopia de Medellín, Colombia / Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia

Resumen Introducción. La claritromicina es el antibiótico de primera línea para el tratamiento de la infección por Helicobacter pylori. La resistencia bacteriana se produce principalmente por mutaciones puntuales del gen ARN ribosómico 23S (ARNr 23S). Objetivo. Determinar la frecuencia de las mutaciones puntuales A2143G y A2142G del gen ARNr 23S asociadas con la resistencia de H. pylori a la claritromicina en muestras de pacientes con manifestaciones dispépticas en Medellín, región noroccidental de Colombia. Materiales y métodos. Se extrajo ADN a partir de muestras de biopsia gástrica obtenidas de pacientes con manifestaciones dispépticas atendidos en una unidad de endoscopia entre el 2016 y el 2017. Mediante reacción en cadena de la polimerasa (PCR), se amplificaron las regiones s y m del gen vacA y una región del gen ARNr 23S bacteriano. La presencia de las mutaciones A2142G y A2143G se determinó por la técnica de polimorfismos de longitud de fragmentos de restricción (RFLP) con las enzimas BbsI y BsaI, respectivamente. Resultados. Se encontró una prevalencia de infección de 44,2 % (175/396), según el informe de histopatología. En 143 de estas 175 muestras positivas se amplificaron las tres regiones del genoma bacteriano. Se identificaron las mutaciones A2143G y A2142G en 27 muestras (18,8 %; 27/143), la mutación más frecuente fue la A2143G (81,5 %; 22/27). Conclusiones. Hubo una gran prevalencia de mutaciones asociadas con la resistencia de H. pylori a la claritromicina en la población de estudio. Se requieren estudios adicionales para establecer la resistencia bacteriana en la población colombiana y, así, determinar los tratamientos de primera línea y de rescate.

Abstract Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.

Resistencia inducible a clindamicina en Staphylococcus aureus resistentes a meticilina aislados de pacientes pediátricos en Paraguay / Inducible resistance to clindamycin in methicillin-resistant Staphylococcus aureus isolated from Paraguayan children

Resumen Introducción: El método de difusión de doble disco se presenta como una alternativa diagnóstica que permite identificar aislados de Staphylococcus aureus susceptibles a clindamicina, ante el aumento de resistencia a meticilina, reduciendo así la posibilidad de fallo en el tratamiento. Objetivo: Determinar la frecuencia de resistencia a clindamicina inducida por eritromicina en S. aureus resistentes a meticilina (SARM) aislados de niños paraguayos. Materiales y Métodos: Estudio observacional, descriptivo, de corte transversal. Se colectaron 145 aislados S. aureus que causaron infecciones de piel y tejidos blandos y osteo-articulares en pacientes pediátricos del Hospital Central del Instituto de Previsión Social en el período de diciembre-2012 a noviembre-2013. La resistencia a clindamicina se determinó por métodos automatizados y de difusión de doble disco. Se realizó reacción de polimerasa en cadena para genes ermA, ermB, ermC y msrA de aislados representativos. Resultados: La resistencia global a meticilina y clindamicina fue de 67 y 13%, respectivamente (11% atribuible al mecanismo de resistencia a clindamicina inducible). Los genes ermC y msrA fueron detectados individualmente en 25 y 17% de los aislados, respectivamente, mientras que un aislado presentó ambos genes en simultáneo. Discusión: La frecuencia de mecanismo de resistencia inducible a clindamicina señala la importancia de los métodos de difusión de doble disco en la práctica microbiológica, así como se encuentran en los límites de puntos de cortes considerados como aceptables para el uso de este antimicrobiano para infecciones cutáneas y osteo-articulares causadas por SARM.

Background: The double disc diffusion method is an alternative diagnostic that allows the identification of Staphylococcus aureus isolates apparently susceptible to clindamycin but that may develop resistance due to an induction phenomena, mainly asociated to the increase in resistance to methicillin, thus increasing the possibility of failure in the treatment. Aim: To determine the frequency of induced clindamycin resistance in methicillin-resistant S. aureus (MRSA) isolated from Paraguayan children. Materials and Methods: In this cross sectional study, we collected 145 S. aureus isolates that caused skin and soft tissue and osteoarticular infections in pediatric patients of the Central Hospital I.P.S. in the period from December-2012 to November-2013. Resistance to clindamycin was determined by automated methods and double disc diffusion. PCR was performed for ermA, ermB, ermC and msrA genes from representative isolates. Results: The global resistance to methicillin and clindamycin was 67 and 13%, respectively (11% attributable to the inducible mechanism). The ermC and msrA genes were detected individually in 25 and 17% of the isolates respectively while an isolate presented both genes simultaneously. Discussion: The frequency of inducible resistance to clindamycin indicates the importance of double disc diffusion methods in microbiological practice, as well as being within the cut off points considered acceptable for the use of this antibiotic for skin infections. and osteoarticular caused by MRSA.

Helicobacter pylori: resistencia múltiple en pacientes de Bogotá, Colombia / Helicobacter pylori: Multiple resistance in patients from Bogotá, Colombia

Resumen Introducción. La resistencia a los antibióticos es la principal causa del fracaso del tratamiento contra Helicobacter pylori; la claritromicina y el metronidazol son los antibióticos que generan mayor resistencia. En Colombia, la resistencia primaria a estos dos antibióticos y el uso excesivo de levofloxacina han alcanzado los límites aceptados (13,6, 83 y 16 %, respectivamente). A pesar de ello, se usa el tratamiento empírico combinando estos antibióticos en pacientes en los que ha fallado anteriormente. Objetivo. Determinar la resistencia a los antibióticos en pacientes previamente tratados para H. pylori en Bogotá, Colombia. Materiales y métodos. Se llevó a cabo un estudio descriptivo en el que se evaluó mediante dilución en agar la resistencia a la amoxicilina, la claritromicina, la levofloxacina y el metronidazol en 10 aislamientos provenientes de 5 pacientes con tres o cuatro tratamientos fallidos para H. pylori. La resistencia a los antibióticos se confirmó mediante secuenciación de ADN (Magrogen, Korea). Resultados. Ocho de los aislamientos presentaron resistencia a dos o más antibióticos y todos fueron resistentes a la levofloxacina. Los patrones de sensibilidad de los aislamientos provenientes del antro pilórico y del cuerpo del estómago, fueron diferentes en tres de los pacientes. Conclusión. Hasta donde se sabe, esta es la primera evidencia de resistencia múltiple de H. pylori en Colombia en pacientes previamente tratados. Los resultados evidenciaron las consecuencias del uso de un esquema ineficaz de tratamiento antibiótico y la necesidad de evaluar la sensibilidad a los antibióticos en diferentes sitios anatómicos del estómago. La resistencia múltiple limita el número de antibióticos útiles para erradicar H. pylori.

Abstract Introduction: The main cause for Helicobacter pylori infection treatment failure is antibiotic resistance, where clarithromycin and metronidazole play the main role. In Colombia, primary resistance as a consequence of the use of these two antibiotics and excessive levofloxacin use is above the accepted limit (13.6%, 83%, and 16%, respectively). Despite this fact, empirical therapies that include the combination of these antibiotics are used in patients with previous therapeutic failure. Objective: To determine antibiotic resistance in patients previously treated for H. pylori in Bogotá, Colombia. Materials and methods: We conducted a descriptive study that included ten isolates obtained from five patients with three or four previous failed treatments for H. pylori. Antibiotic resistance to amoxicillin, clarithromycin, levofloxacin, and metronidazole was investigated by agar dilution and confirmed by DNA sequencing (Magrogen, Korea). Results: Eight isolates were resistant to two or more antibiotics. All isolates were resistant to levofloxacin. Susceptibility patterns in isolates from the gastric antrum and the body of the stomach were different in three patients. Conclusion: As far as we know, this is the first evidence of multiple H. pylori resistance in Colombia in previously treated patients. Results demonstrated the consequences of using an ineffective antibiotic scheme and the need to assess antibiotic susceptibility in different anatomical sites of the stomach. The consequences of multiple resistance decrease possible antibiotic effectiveness to eradicate H. pylori in the future.

Detection of multidrug-resistant Enterobacteriaceae isolated from river waters flowing to the Guanabara Bay and from clinical samples of hospitals in Rio de Janeiro, Brazil / Detección de enterobacterias multirresistentes aisladas en aguas de los ríos que desembocan en la bahía de Guanabara y en muestras de hospitales de Río de Janeiro, Brasil

Abstract Introduction: The use of antibiotics in humans, animal husbandry and veterinary activities induces selective pressure leading to the colonization and infection by resistant strains. Objective: We evaluated water samples collected from rivers of the Guanabara Bay, which have suffered minor and major environmental degradation, and clinical samples of hospital origin to detect evidence of the presence of resistance genes to aminoglycosides, beta-lactam antibiotics and fluoroquinolones in strains of Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and Escherichia coli. Materials and methods: For isolation of the water strains we employed culture media containing 32 μg/ml cephalotin and 8 μg/ml gentamicin. The strains from clinical materials were selected using culture media containing 8 μg/ml gentamicin. The strains were identified and subjected to antimicrobial susceptibility testing (AST), plasmid DNA extraction and polymerase chain reaction (PCR) to detect genes encoding enzymes modifying aminoglycosides (EMA), extended-spectrum beta-lactamases (ESBL) and plasmid mechanisms of quinolone resistance (PMQR). Results: The AST of the isolates recovered from water samples showed multidrug-resistance profiles similar to those found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates from clinical samples showed at least one plasmid band. In the PCR assays, 7.4% of the isolates recovered from water samples and 20% of those from clinical materials showed amplification products for the three antimicrobial classes. Conclusion: We believe that the detection of microorganisms presenting genetic elements in environments such as water is necessary for the prevention and control of their dissemination with potential to infect humans and other animals in eventual contact with these environments.

Resumen Introducción. El uso de antibióticos en seres humanos, en la industria pecuaria y en las actividades veterinarias induce una presión selectiva que resulta en la colonización e infección con cepas resistentes. Objetivo. Determinar la presencia de genes de resistencia a aminoglucósidos, betalactámicos y fluoroquinolonas en cepas de Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae y Escherichia coli, obtenidas de muestras de agua de los ríos que desembocan en la bahía de Guanabara y de muestras clínicas de hospitales de Río de Janeiro. Materiales y métodos. En la selección de las cepas resistentes obtenidas de las muestras de agua de los ríos, se emplearon medios de cultivo que contenían 32 μg/ml de cefalotina y 8 μg/ ml de gentamicina. En el caso de las muestras de especímenes clínicos, se usaron medios de cultivo que contenían 8 μg/ml de gentamicina. Las cepas se identificaron y se sometieron a pruebas de sensibilidad antimicrobiana, extracción de ADN plasmídico y pruebas de reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican aquellas enzimas que modifican los aminoglucósidos, las betalactamasas de espectro extendido (BLEE) y los mecanismos de resistencia a las quinolonas mediados por plásmidos. Resultados. Se encontraron perfiles de resistencia a los antimicrobianos similares en los dos grupos. En todas las bacterias obtenidas de las muestras de agua y en 90 % de las muestras clínicas, se evidenciaron bandas de plásmidos asociados con la transferencia de genes de resistencia. En las pruebas de PCR, se obtuvieron productos de amplificación de los genes de resistencia para las tres clases de antimicrobianos analizados, en el 7,4 % de las bacterias recuperadas de las muestras de agua y en el 20 % de aquellas recuperadas de las muestras clínicas. Conclusión. La detección de microorganismos con elementos genéticos que confieren resistencia a los antibióticos en ambientes como el agua, es una estrategia necesaria para prevenir y controlar la diseminación de estos agentes patógenos con potencial para infectar a humanos y a otros animales en dichos ambientes.

Distribución y caracterización molecular de betalactamasas en bacterias Gram negativas en Colombia, 2001-2016 / Distribution and molecular characterization of beta-lactamases in Gram-negative bacteria in Colombia, 2001-2016

Resumen Las betalactamasas, enzimas con capacidad hidrolítica frente a los antibióticos betalactámicos, son responsables del principal mecanismo de resistencia en bacterias Gram negativas; las de mayor impacto clínico y epidemiológico en los hospitales, son las betalactamasas de espectro extendido (BLEE), las de tipo AmpC y las carbapenemasas. El incremento en su frecuencia y su diseminación a nivel mundial ha limitado cada vez más las opciones terapéuticas tanto en infecciones adquiridas en los hospitales como las que se generan en la comunidad. En Colombia, las redes de vigilancia y los grupos de investigación iniciaron su estudio desde finales de los años 90 y, así, se logró la caracterización molecular de las diferentes variantes; además, se reportó una gran prevalencia y diseminación en los hospitales de mediana y alta complejidad, y se describió el impacto clínico de las infecciones que causan. Dichos estudios han evidenciado el alto grado de endemia de algunas de estas betalactamasas y, en consecuencia, la necesidad de una inmediata implementación de programas para inducir el uso prudente de los antibióticos y de medidas de vigilancia, que permitan controlar y prevenir su diseminación, con el fin de disminuir la morbimortalidad en los pacientes y preservar las opciones terapéuticas disponibles en la actualidad. En esta revisión, se recopiló la información sobre las variantes, la distribución geográfica y la caracterización molecular de las betalactamasas en Colombia, así como los estudios llevados a cabo desde finales de la década de 90 hasta el 2016, lo cual permitió tener un panorama de las betalactamasas que circulan en diferentes regiones, su incremento en el tiempo y sus implicaciones clínicas.

Abstract Beta-lactamases are enzymes with hydrolytic activity over beta-lactam antibiotics and they are the main resistance mechanism in Gram-negative bacteria. Extended-spectrum beta-lactamases (ESBL), AmpC, and carbapenemases have the greatest clinical and epidemiological impact in hospital settings. The increasing frequency and worldwide spread of these enzymes have limited the therapeutic options in hospital-acquired infections and those originating in the community. In Colombia, surveillance networks and research groups began studying them in the late 90s. Different variants of these enzymes have been molecularly characterized and their high prevalence and dissemination in medium and high complexity hospitals, along with a high clinical impact, have been reported. Furthermore, many studies in Colombia have evidenced high endemicity for some of these beta-lactamases, which requires an urgent implementation of antimicrobial stewardship programs in order to preserve the few therapeutic options and infection control strategies to prevent and limit their dissemination. In this publication, we carried out a review of the different enzyme variants, geographic distribution, and molecular characterization of these beta-lactamases in Colombia. Additionally, we describe the available information in the literature regarding studies conducted between the late 1990s and 2016, which provide an overview of the beta-lactamases circulating in different regions of Colombia, their increase over time, and their clinical implications.

Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin / 中国中药杂志

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Rapid, specific, and sensitive detection of the ureR_1 gene in Klebsiella pneumoniae by loop-mediated isothermal amplification method

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae) / Filogenia do endosimbionte Wolbachia em Trichogramma, an alpha-proteobacteria (Rickettsiae)

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".

Caracterização de Escherichia coli enteroagregativa entre crianças com diarreia na Amazônia ocidental brasileira / Characterization of enteroaggregative escherichia coli among diarrheal childrenin western brazilian amazon

ABSTRACT BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries. OBJECTIVE: The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State. METHODS: The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method. RESULTS: The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant. CONCLUSION: As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.

RESUMO CONTEXTO: A Escherichia coli enteroagregativa (EAEC) é um dos principais agentes causadores de diarreia aguda e crônica em crianças e adultos, principalmente em países em desenvolvimento. OBJETIVO: Caracterizar cepas de EAEC isoladas de amostras fecais e identificar genes que potencialmente contribuem para a virulência, produção de biofilme e resistência antimicrobiana em crianças internadas em um hospital pediátrico em Porto Velho, Rondônia. MÉTODOS: Um total de 1.625 cepas de E. coli foram isolados de 591 crianças com gastroenterite aguda na faixa etária de 6 anos que foram internadas no Hospital Infantil Cosme e Damião na cidade de Porto Velho, entre fevereiro de 2010 e fevereiro de 2012. Colônias sugestivas de E. coli foram submetidas a reação em cadeia da polimerase para identificação de fatores de virulência. O ensaio de adesão in vitro foi desenvolvido com célula HEp-2. A detecção de biofilme foi realizada através do teste de espectrofotometria e os testes de susceptibilidade aos antimicrobiana foram realizados através do método de difusão em disco. RESULTADOS: A E. coli diarreiogênica foi encontrada em 27,4% (162/591) das crianças e a EAEC foi a E. coli diarreiogênica mais frequentemente associada à diarreia com 52,4% (85/162), seguida pela E. coli enteropatogênica 43,8% (71/162), E. coli enterotoxigênica 2,4% (4/162) e E. coli enterohemorrágica 1,2% (2/162). O gene aggR foi detectado em 63,5% (54/85) dos isolados de EAEC com correlação estatisticamente significante entre esse gene com os genes aatA (P<0,0001), irp2 (P=0,0357) e shf (P=0,0328). Neste estudo 69% (59/85) das cepas de EAEC eram produtoras de biofilme, destas 73% (43/59) possuíam o gene aggR, ao passo que entre as não produtoras 42,3% (11/26) possuíam o gene (P=0,0135). Essa associação também foi observada com o gene aatA, presente em 61% (36/59) das cepas produtoras e em 19,2% (5/26) das não produtoras (P<0,0004). O teste de sensibilidade aos antibimicrobianos evidenciou que a maioria das EAEC eram resistentes a ampicilina 70,6% (60/85), ao sulfametoxazol 60% (51/85), a tetraciclina 44,7% (38/85) e a cefotaxima 22,4% (19/85). CONCLUSÃO: Este é o primeiro estudo no Norte do Brasil sobre a investigação dos fatores de virulência de EAEC mostrando a susceptibilidade antimicrobiana de cepas de EAEC isoladas de crianças com diarreia.

Multidrug-resistant Acinetobacter baumannii clones persist on hospital inanimate surfaces

ABSTRACT Acinetobacter baumannii is one of the most frequent Gram-negative opportunistic pathogens associated with hospital-acquired infection worldwide. We briefly describe A. baumannii isolates that were recovered from surrounding ICU bed surfaces, exhibiting multidrug resistance phenotype and belonging to some widely spread clonal complexes of clinical A. baumannii isolates.