[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 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.

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.

RpoS integrates CRP, Fis, and PhoP signaling pathways to control Salmonella Typhi hlyE expression.

BACKGROUND: SPI-18 is a pathogenicity island found in some Salmonella enterica serovars, including S. Typhi. SPI-18 harbors two ORFs organized into an operon, hlyE and taiA genes, both implicated in virulence. Regarding the hlyE regulation in S. Typhi, it has been reported that RpoS participates as transcriptional up-regulator under low pH and high osmolarity. In addition, CRP down-regulates hlyE expression during exponential growth. Previously, it has been suggested that there is another factor related to catabolite repression, different from CRP, involved in the down-regulation of hlyE. Moreover, PhoP-dependent hlyE up-regulation has been reported in bacteria cultured simultaneously under low pH and low concentration of Mg2+. Nevertheless, the relative contribution of each environmental signal is not completely clear. In this work we aimed to better understand the regulation of hlyE in S. Typhi and the integration of different environmental signals through global regulators. RESULTS: We found that Fis participates as a CRP-independent glucose-dependent down-regulator of hlyE. Also, Fis and CRP seem to exert the repression over hlyE through down-regulating rpoS. Moreover, PhoP up-regulates hlyE expression via rpoS under low pH and low Mg2+ conditions. CONCLUSIONS: All these results together show that, at least under the tested conditions, RpoS is the central regulator in the hlyE regulatory network, integrating multiple environmental signals and global regulators.

Risk factors for prolonged hospitalization after gynecologic laparoscopic surgery.

OBJECTIVE: To determine perioperative risk factors for prolonged hospitalization after gynecologic laparoscopic surgery. METHODS: Data on patients who underwent gynecologic laparoscopic surgery at a single academic institution from January 2000 to January 2009 were evaluated. Patient demographics, clinical history, intraoperative data, and postoperative adverse events were analyzed. Logistic regression analysis identified significant predictors of prolonged hospitalization (hospital stay>48 h after surgery). A risk score was created from the analysis to predict prolonged hospitalization. RESULTS: Eight hundred seven patients were included. The median body mass index was 26.5 kg/m(2) (range, 14.2-72.3 kg/m(2)), and the median age was 49 years (range, 12-88 years). Four hundred fifty-nine patients (56.9%) underwent surgery for benign conditions, and 348 (43.1%) underwent surgery for malignant disease. A total of 78 patients (9.7%) had a prolonged hospitalization. Independent predictors of prolonged hospitalization were age>54 years (P<0.0001), operative blood loss>120 mL (P<0.0001), intraoperative or postoperative blood transfusion (P=0.0237), and early postoperative complication (P<0.0001). Having a prior laparoscopy was associated with a shorter hospital stay (P=0.0276). The risk score showed how changes in perioperative factors change the risk of prolonged hospitalization. CONCLUSION: Factors such as age, blood loss, perioperative blood transfusion, and postoperative complications are associated with prolonged length of stay after laparoscopic surgery, while having a prior laparoscopy is associated with a shorter hospital stay. A clinical scoring system can be used to estimate probability of prolonged hospitalization after gynecologic laparoscopic surgery.

The carbon source influences the efflux pump-mediated antimicrobial resistance in clinically important Gram-negative bacteria.

OBJECTIVES: Multidrug efflux pumps are proteins known to play an important role in resistance in bacteria. These proteins are located in the inner membrane (IM), together with many other proteins, including inducible permeases that participate in the uptake of non-phosphotransferase system (PTS) carbohydrates (i.e. carbohydrates uptaken by mechanisms other than the PTS). However, lipid bilayer space in the IM is limited. Therefore, we examined whether the overexpression of unrelated IM proteins is able to interfere with the efflux-mediated resistance mechanism, consequently increasing the susceptibility towards different antimicrobial compounds. METHODS: We cultured bacteria under different conditions that increase the synthesis of unrelated IM proteins, either by using a non-PTS carbohydrate as the sole carbon source or by artificially overexpressing IM proteins, prior to determining the resistance to different antimicrobial compounds by disc diffusion assays. RESULTS: We observed that efflux-pump-mediated resistance is affected by the carbon source in all the strains tested, exhibiting increased susceptibility when a non-PTS carbohydrate was used as the sole carbon source. Moreover, when we artificially overexpressed an unrelated IM protein, we also observed decreased efflux-mediated resistance. CONCLUSIONS: These results strongly suggest that overexpression of IM proteins, by using a non-PTS carbohydrate as the sole carbon source, or by artificially introducing a high number of copies of an unrelated IM protein, competes with the antibiotic efflux systems, thereby decreasing the efflux-mediated resistance to different antimicrobial compounds. This sort of competition arises because of the limited available space in the bacterial IM, or by an unknown mechanism.

RpoS- and Crp-dependent transcriptional control of Salmonella Typhi taiA and hlyE genes: role of environmental conditions.

A novel pathogenicity island, SPI-18, carries the taiA-hlyE operon, encoding virulence factors in Salmonella Typhi. To determine the effects of certain environmental conditions on the expression of these genes, beta-galactosidase assays, RT-PCR reactions, western blot analyses and measurement of hemolytic activity were performed. The conditions studied are those likely found by S. Typhi during infection in the human host. We found RpoS-dependent transcriptional upregulation in low pH and high osmolarity for both genes. Our results show that oxygen depletion apparently did not affect transcription of the taiA-hlyE operon. On the other hand, the transcriptional regulator Crp, previously described as an activator of hlyE transcription in Escherichia coli, is involved in transcriptional repression of hlyE in S. Typhi. Moreover, addition of glucose to the growth medium results in decreasing the hlyE mRNA, suggesting that there is another factor related to catabolite repression different from Crp and involved in downregulation of hlyE in S. Typhi.