Salmonella enterica serovar Typhi plasmid pR ST98 -mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts.

pR ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST 8 carrying pR ST98 , Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST 10 without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST 8 containing pR ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR ST98 -mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR ST98 -mediated virulence in S. typhi.

Salmonella enterica serovar typhi plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy

OBJECTIVES: Plasmid pR ST98 is a hybrid resistance-virulence plasmid isolated from Salmonella enterica serovar Typhi (S. typhi). Previous studies demonstrated that pR ST98 could enhance the virulence of its host bacteria. However, the mechanism of pR ST98-increased bacterial virulence is still not fully elucidated. This study was designed to gain further insight into the roles of pR ST98 in host responses. METHODS: Human-derived macrophage-like cell line THP-1 was infected with wild-type (ST8), pR ST98-deletion (ST8-ΔpR ST98), and complemented (ST8-c-pR ST98) S. typhi strains. Macrophage autophagy was performed by extracting the membrane-unbound LC3-I protein from cells, followed by flow cytometric detection of the membrane-associated fraction of LC3-II. Intracellular bacterial growth was determined by colony-forming units (cfu) assay. Macrophage cell death was measured by flow cytometry after propidium iodide (PI) staining. Autophagy activator rapamycin (RAPA) was added to the medium 2 h before infection to investigate the effect of autophagy on intracellular bacterial growth and macrophage cell death after S. typhi infection. RESULTS: Plasmid pR ST98 suppressed autophagy in infected macrophages and enhanced intracellular bacterial growth and S. typhi-induced macrophage cell death. Pretreatment with RAPA effectively restricted intracellular bacterial growth of ST8 and ST8-c-pR ST98, and alleviated ST8 and ST8-c-pR ST98-induced macrophage cell death, but had no significant effect on ST8-ΔpR ST98. CONCLUSIONS: Plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy.

Correlation studies on Widal agglutination reaction and diagnosis of typhoid fever.

A total of 80 patients at the University of Uyo Teaching Hospital (UUTH) suspected of having enteric infections were screened for the presence of Salmonella species using blood, urine and stool samples along with Widal agglutination tests. Although 39 patients tested positive for the Widal agglutination test with titers ranging from 1:80 to 1:320, no Salmonella organism was encountered in some cultures. Statistical analysis revealed significant differences (chi2) at the 5% probability level between the Widal test and the cultures of the clinical samples. The results suggest that serological investigations alone may not be a reliable index for the diagnosis of Salmonella infections.

A propósito de dos casos de fiebre tifoidea: valor diagnóstico de la prueba de Widal y uso de ciprofloxacina como tratamiento de elección / A purpose two cases of typhoid fever: diagnostic value of the widal test and the use of aprofloxacina as first choice treatment discussed

Se presentan dos casos de síndrome febril de más de 7 días de evolución, en los que se demostró infección por Salmonella typhi. El diagnóstico de fiebre tifoidea se logró temporalmente por hemocultivos. La clínica en todo momento fue inespecífica. Se discute el uso y utilidad clínica de la prueba Widal, así como el empleo de ciprofloxacina como tratamiento de elección para esta infección

Comparación de tres métodos empleados para el control de calidad de medios de cultivo sólido / Comparison of three methods employed in tue quality control of solid culture media

El objetivo de este estudio fue comparar tres métodos utilizados en el control de calidad de medios de cultivo: a)el método original de Miles-Mirsa; b)el ecométrico y c)el método de estrías. Para comparar estos métodos, se evaluaron la productividad (crecimiento de un microorganismo que habitualmente se desarrolla en un medio de cultivo) y la selectividad (supresión del crecimiento de un microorganismo, que se espera sea inhibido en un medio de cultivo) de seis medios sólidos de cultivo usados para el aislamiento de Salmonella spp. y Shigella spp. Los resultados que se obtuvieron al comparar los 3 métodos, mediante la prueba de Chi2, reflejan que existe diferencia significativa (p<0.05) entre el método de estrías y los otros 2 en lo referente a la productividad, no existiendo diferencia significativa entre los 3 métodos, en cuanto a selectividad. De los 3 métodos ensayados, el ecométrico resultó el de más fácil y rápida ejecución

Osteomielitis vertebral salmonelósica. Informe de dos casos / Salmonella vertebral osteomyelitis. Report of two cases

La osteomielitis salmonelósica es muy rara, con una incidencia de 0.45%de todas las osteomielitis. Se considera que sólo 0.8%de los casos de fiebre tifoidea, desarrollarán infección ósea. Comunicamos dos casos de osteomielitis vertebral por Salmonella typhi que fueron atendidos en el Hospital de Traumatología y Ortopedia Lomas Verdes del IMSS. El primer caso, hombre de 55 años de edad, con infección salmonelósica de seis meses de evolución y que recibió tratamiento antibacteriano con cloramfenicol y ampicilina; localización osteomielítica vertebral torácica, T9-T10, con paraplejia y deterioro general, en el cual se efectuó descompresión medular con resección-biopsia, artrodesis intersomática por toracotomía y en un segundo tiempo instrumentación de Luque. El cultivo de la muestra operatoria mostró desarrollo de Salmonella typhi resistente a todos los antibacterianos del cuadro básico y en el cual se utilizaron quinolonas con buena respuesta terapéutica. El segundo caso, hombre de 19 años de edad, con lumbalgia y cuadro febril de tres meses de evolución, con localización osteomielítica vertebral lumbar L4, sin compromiso neurológico, en el cual el diagnóstico se estableció con la positividad de la reacción de Widal, utilizándose tratamiento antibacteriano con quinolonas y reposo en cama. En ambos, la evolución fue hacía la curación con desaparición del compromiso neurológico en el paciente que lo presentaba.

The use of a coagglutination test to presumptively identify Salmonella typhi in bone marrow-oxgall medium cultures from typhoid fever patients.

A study was conducted to test a coagglutination procedure for detection of Salmonella typhi in bone marrow cultures from suspected typhoid patients admitted to Friendship Hospital, Jakarta, Indonesia. The results of the coagglutination tests were compared to the results from standard cultural isolation and identification. Bone marrow aspirates (356) were cultured in oxgall medium and aliquots subcultured daily for 7 days while simultaneously testing for the presence of Salmonella group D and Vi antigens using coagglutination (COAG). S. typhi was isolated from 220 (62%) of the cultures and the D- and Vi-COAG tests were positive for those same cultures. The COAG test was also negative for 6 cultures containing S. paratyphi A. The COAG results were available within 10 minutes after 18 to 24 hours incubation of the primary cultures whereas the isolation and confirmed identification took 2 to 3 days longer. The COAG test is valuable as an aid to rapidly identify S. typhi in bone marrow-oxgall cultures.

Evaluation of thiol broth for the culture of Salmonella typhi and other bacteria from blood

Thiol broth is known to neutralize various antimicrobial agents. Positivity of growth of various species of bacteria from blood in thiol broth was reported as similar to that in tryptic soy broth (TSB). As blood cultures are often used for the diagnosis of typhoid fever, and as patients may receive antimicrobial therapy before blood culture, the positivity and rapidity of growth of Salmonella typhi in thiol broth were compared to those in TSB. Routine blood culture samples from Yonsei Medical Center patients were inoculated in 50-ml amounts of TSB and thiol broth. The media were prepared from dehydrated products and did not contain CO2, but TSB contained 0.025% sodium polyanethol sulfonate (SPS). Growth of S. paratyphi-A, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens and alpha-hemolytic Streptococcus were similar in both media. However, greater positivity and shorter incubation time for macroscopic detection were noted in TSB with S. typhi, Escherichia coli and Staphylococcus aureus. It is concluded that thiol broth is inferior to TSB plus SPS for the culture of S. typhi from blood.

Effect of Hypertonic Sucrose on the Growth of Salmonella typhi in Experimental Blood Cultures

Slower growth of S. typhi in hypertonic media, reported previously by the authors, was contradictory to other workers', results which showed better growth of some species of bacteria. To evaluate furthur the effect of hypertonic sucrose on the growth of S. typhi, organisms were suspended in saline or in blood with or without sodium polyanethol sulfonate (SPS) and stored up to 24 hours. And then viable counts were determined on tryptic soy agar (TSA) and experimental blood cultures were done in tryptic soy broth (TSB) and in TSB with 10% sucrose (TSB-H). S. typhi, suspended in blood and kept for 24 hours, were inoculated into TSB and TSB-H and after 4 hour incubation viable counts were made on TSA and on TSA with 10% sucrose (TSA-H). In this study it was found that, during the 24 hour storage, the viable counts of S. typhi suspended in saline with or without SPS were similar and those suspended in blood with SPS were incereasing. Comparison of the growth in TSB and in TSB-H did not show hyperonic media was better for the cultivation of S. thphi which was kept up to 24 hours before inoculation. On the contrary the growth was slower. Viable counts made on TSA and on TSA-H from the TSB and TSB-H, which were inoculated with S. typhi suspended in blood and incubated for 4 hours, showed similar results indicating TSB-H did not support faster growth. From the results of this experiment and of the previous clinical blood cultures, it is concluded that 0.1% SPS does not give adverse effect on S. typhi during the 24 hour storage and that hypertonic sucrose does not give better result in the cultivation of S. typhi.

Effects of High Concentrations of Sucrose in Blood Culture Media with Special Reference to the Cultivation of Salmonella typhi

Osmotically stabilized media have been reported to increase the recovery rate of various bacteria from blood. This study was made to determine the effect of high concentrations of sucrose on the cultivation of S. typhi from blood. Sucrose in 15% or 30% concentration in the blood culture media retarded the growth. The mean incubation time for the appearance of growth was significantly longer in the media with sucrose. In those blood specimens which rendered growth of S. typhi in both media with and without sucrose, the incubation times were compared; and it was found that the majority of the specimens showed faster growth in the media without sucrose. Experimental cultures showed that the higher the sucrose concentration the lighter and slower were the growths of S. typhi. These tendencies were also observed in the growth of E. coli, P. aeruginosa, S. aureus, beta-hemolytic Streptococcus, alpha-hemolytic Streptococcus and S. pneumoniae.