stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte--bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Δstg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukaryotic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

Isolation of a novel bacteriophage specific for the periodontal pathogen Fusobacterium nucleatum.

Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. We isolated a new bacteriophage (FnpΦ02) that specifically infects this bacterium. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a segmented tail. The size of the phage genome was estimated to be approximately 59 kbp of double-stranded DNA. The morphological features and the genetic characteristics suggest that FnpΦ02 is part of the Siphoviridae family. Using one-step growth and adsorption experiments, the latent period, burst size, and adsorption rate were estimated to be 15 h, 100 infectious units per cell, and 7.5 × 10⁻¹° ml min⁻¹, respectively. A small fragment of phage DNA was cloned and sequenced, showing 93% nucleotide identity with the phage PA6 of Propionibacterium acnes and amino acid identity with fragments of two proteins (Gp3 and Gp4) of this phage. To our knowledge, FnpΦ02 is the first phage described to infect Fusobacterium nucleatum and provides the base for future exploration of phages in the control of periodontal disease.

S. Typhimurium sseJ gene decreases the S. Typhi cytotoxicity toward cultured epithelial cells.

BACKGROUND: Salmonella enterica serovar Typhi and Typhimurium are closely related serovars as indicated by >96% DNA sequence identity between shared genes. Nevertheless, S. Typhi is a strictly human-specific pathogen causing a systemic disease, typhoid fever. In contrast, S. Typhimurium is a broad host range pathogen causing only a self-limited gastroenteritis in immunocompetent humans. We hypothesize that these differences have arisen because some genes are unique to each serovar either gained by horizontal gene transfer or by the loss of gene activity due to mutation, such as pseudogenes. S. Typhi has 5% of genes as pseudogenes, much more than S. Typhimurium which contains 1%. As a consequence, S. Typhi lacks several protein effectors implicated in invasion, proliferation and/or translocation by the type III secretion system that are fully functional proteins in S. Typhimurium. SseJ, one of these effectors, corresponds to an acyltransferase/lipase that participates in SCV biogenesis in human epithelial cell lines and is needed for full virulence of S. Typhimurium. In S. Typhi, sseJ is a pseudogene. Therefore, we suggest that sseJ inactivation in S. Typhi has an important role in the development of the systemic infection. RESULTS: We investigated whether the S. Typhi trans-complemented with the functional sseJ gene from S. Typhimurium (STM) affects the cytotoxicity toward cultured cell lines. It was found that S. Typhi harbouring sseJSTM presents a similar cytotoxicity level and intracellular retention/proliferation of cultured epithelial cells (HT-29 or HEp-2) as wild type S. Typhimurium. These phenotypes are significantly different from wild type S. Typhi CONCLUSIONS: Based on our results we conclude that the mutation that inactivate the sseJ gene in S. Typhi resulted in evident changes in the behaviour of bacteria in contact with eukaryotic cells, plausibly contributing to the S. Typhi adaptation to the systemic infection in humans.

[Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli]. / Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes.

Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes / Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli

Resumen Utilizando cepas clínicas de bacilos gramnegativos multi-resistentes (MDR), comparamos las CIM obtenidas de la microdilución en caldo, el método de referencia y el método de elución de sensidiscos. Encontramos que, con la excepción de A. baumannii, los resultados fueron muy similares. El método de elución de sensidiscos podría ser una buena alternativa y confiable para la determinación de la resistencia a colistín.

Abstract Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Identification of bacterial and fungal species in human cadavers used in anatomy teaching / Identificación de especies bacterianas y fúngicas en cadáveres humanos utilizados en la enseñanza de la anatomía

Within the framework of undergraduate and postgraduate medical education, cadavers have been used to teach anatomy by dissection or by using prosected specimens. To accomplish this, an appropriated preservation process must guarantee that the cadaver is kept safe for harm, destruction, and decomposition. Embalming fluid contains fixatives, disinfectants, surfactants, buffers, salt, and water, making the cadaver safe for teaching anatomy. However, it remains unclear if there is any risk of dissemination of microorganisms during anatomy teaching, research, and dissection procedures on fixed cadavers. The purpose of this study is to identify bacterial and fungal species in fixed cadaveric material used in anatomy teaching. Samples of cadavers and anatomical sections were cultured and biochemical tests and molecular identification by polymerase chain reaction (PCR) were performed to identify the microorganisms. The results indicate that fixed cadaveric material has viable bacteria on its surfaces and almost all these correspond to gram-negative bacilli of the Enterobacteriaceae family. In conclusion, fixed cadavers could be a reservoir of bacteria. This study underscores the importance of generating safe manipulation protocols to avoid eventual contamination and disease.

Dentro del curriculum de los programas de postgrado y pregrado de las carreras de la salud, los cadáveres han sido utilizados para la enseñanza de la anatomía mediante la disección o utilizando preparados anatómicos. Para poder llevar a cabo esto, el cadáver debe pasar por un adecuado proceso de preservación; en el que se utilizan fluidos que contienen fijadores, desinfectantes, surfactantes, buffers, sal y agua, los cuales lo protegen del deterioro y la descomposición. Las soluciones fijadoras y conservadoras contienen desinfectantes, surfactantes, fijadores, buffers, sal y agua, que hacen que el cadáver sea seguro para la enseñanza de la anatomía. Sin embargo, no está claro si existe algún riesgo de diseminación de microorganismos durante la enseñanza, investigación y/o disección en estos cadáveres. El propósito del estudio es identificar especies bacterianas y/o fúngicas en material cadavérico previamente fijado, usado en la enseñanza de la anatomía. Se realizaron cultivos y técnicas de identificación molecular mediante reacción en cadena de polimerasa de muestras tomadas desde material cadavérico para identificar los microorganismos encontrados. Los resultados indican que el material cadavérico previamente fijado posee bacterias en sus superficies, la mayoría corresponde a bacilos gram negativos de la familia de las Enterobacteriaceae. En conclusión, los cadáveres previamente fijados pueden ser reservorio de bacterias. Este estudio destaca la importancia de generar protocolos de manipulación con el fin de evitar una posible contaminación y enfermedad.

stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte-bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Astg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukary-otic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.