Comparison of effects of staphylococcal nuclease A fused with different exogenous DNA fragments / 生物工程学报

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Mutation of lambdapL/pR-cI857 system for production of bacterial ghost in Escherichia coli / 生物工程学报

Bacterial ghost is intact envelope of Gram-negative bacteria, which is produced by the function of the lysis gene E from bacteriophage PhiX174. The expression of the lysis gene E is usually controlled by the thermosensitive lambdapL/pR-cI857 promoter. In this study, we described a mutation (T --> C) at the ninth nucleotide of the OR2 in the lambdapR promoter of the lambdapL/pR-cI857 system by overlap PCR. The bacteriolytic assay showed that the mutation in the lambdapL/pR-cI857 system enhanced the temperature of repressing the expression of gene E up to 37 degrees C. The lysis efficiency of altered lambdapR promoter in Escherichia coli DH5a and avian pathogenic E. coli DE17 was up to 99.9%. The expanded range of temperature will benefit for the production of bacterial ghost.

Comparative study of two bacteriocins produced by representative indigenous soil bacteria

The aim of this research work was to identify and characterized the bacteriocins produced by soil-associated bacteria. Bacillocin from Bacillus brevis Bb and pyocin from Pseudomonas aeruginosa Pa were found bioactive only against gram-positive bacteria tested. Maximum production of both the bacteriocins was observed at 32°C in BHI medium. Production of both the bacteriocins started in the early exponential growth phase while the maximum production was observed during the stationary phase. Bacillocin Bb remained stable during 1-9 pH while pyocin Pa remained stable at pH 1-11. Both of the bacterocins were found resistant to high temperature [100°C for 30 min], detergents [1% solutions of EDTA, Tween 20, Tween 80 and SDS] and organic solvents [1% solutions of Ethanol, Butanol, Methanol, Propanol, Chloroform, and Acetone]. Activity of both was completely lost after proteinase K treatment suggesting their protein nature. Titre of bacillocin Bb was estimated to be 5280 AU/mL while the titre for pyocin Pa was calculated as 640 AU/mL. Both of the bacteriocins showed bacteriolytic mode of action against the indicator Bacillus strain BC31 and were found <10 KDa in their molecular mass

Expression, purification and characterization of bacteriophage lysin of Streptococcus in Escherichia coli / 生物工程学报

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette.

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

Inhibitory effect of egg white lysozyme on ceftazidime-induced release of endotoxin from Pseudomonas aeruginosa / 药学学报

<p><b>AIM</b>To investigate the inhibitory effect of egg white lysozyme (LZM) on ceftazidime (CFT)-induced release of endotoxin from Pseudomonas aeruginosa.</p><p><b>METHODS</b>P. aeruginosa PAO1 was inoculated in nutrition broth or diluted rabbit blood free of antibiotics in the presence or absence of LZM and incubated at 37 degrees C on a water bath shaker. beta-Lactam antibiotic, CFT, was added to cultures at 3.5 h (nutrition broth culture) or 5 h (diluted rabbit blood culture) after inoculation. After 3 h of CFT treatment, the supernatants from different bacterial cultures were prepared by centrifuge and the concentrations of endotoxin in the supernatants were measured. The bacterial supernatants were also added to a murine macrophage cell line RAW 264.7 or intravenously injected into carrageenin-sensitized mice. Tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) concentrations in RAW 264.7 supernatants or in mouse sera were tested.</p><p><b>RESULTS</b>CFT treatment alone obviously inhibited the growth of P. aeruginosa PAO1 accompanied by strong and rapid bacteriolysis and released relatively high concentration of endotoxin from bacteria both in nutrition broth and in diluted rabbit blood cultures. The bacterial supernatant from CFT treatment alone yielded high concentrations of TNF alpha both in RAW 264.7 cells and in mice and high level of NO in RAW 264.7 cells. Treatment with the combination of LZM and CFT evidently blocked the lysis of bacteria and reduced the release of endotoxin without decreasing bactericidal activity of CFT. TNF alpha and NO productivity of the supernatants prepared from the LZM/CFT combinative treated bacterial cultures were significantly decreased both in RAW 264.7 cells and in mice indicating that the inflammatory activity was reduced.</p><p><b>CONCLUSION</b>LZM can effectively prevent CFT-induced bacteriolysis, endotoxin release and subsequent proinflammatory factor production but without decreasing bactericidal activity of CFT, resulting in the disassociation of bactericidal activity and bacteriolysis. Thus, LZM might be important for preventing endotoxemia in Gram-negative sepsis with the treatment of antibiotics.</p>

Antibacterial effect of polyphosphate on endodontopathic bacteria / 대한치과보존학회지

This study was performed to observe the antibacterial effect of polyphosphate (polyP) with various chain lengths (P3~P75) on virulent, invasive strains of P. gingivalis A7A1-28 and W50, and multidrug resistant E. faecalis ATCC29212. P. gingivalis strains were grown in brain-heart infusion broth (BHI) containing hemin and vitamin K with or without polyP. PolyP was added at the very beginning of the culture or during the exponential growth phase of the culture. Inhibition of the growth of P. gingivalis was determined by measuring the absorbancy at 540nm of the grown cells. Viable cell counts of the culture and release of intracellular nucleotide from P. gingivalis were measured. E. faecalis was grown in plain BHI with antibiotics alone or in combination with polyP(calgon; 0.1~1.0%) and the bacterial absorbancy was measured. The overall results suggest that polyP has a strong antibacterial effect on the growth of the virulent strains of P. gingivalis and the antibacterial activity of polyP seems largely bactericidal, accompanying bacteriolysis in which chelation phenomenon is not involved. Although polyP does not exert antibacterial activity against E. faecalis, it appears to increase antibacterial effect of erythromycin and tetracycline on the bacterium. Therefore, polyP alone or in combination with antibiotics may be developed as a candidate for the agent controlling oral infections including endodontic infection.

Effect of dexamethasone on the inflammation and TNF alpha in experimental rabbit pneumococcal meningitis

It is generally believed that tumor necrosis factor alpha (TNF a) plays an crucial role in the pathogenesis of bacterial meningitis through various mechanisms such as endothelial damage, induction of adhesion molecule on the endothelial cell, recruitment of leukocytes in the cerebrospinal fluid(CSF). We performed this study to analyze the relationship of TNF alpha level with the severity of inflammation in the CSF. In a rabbit meningitis model, we attempted to evaluate whether antibiotics induced bacteriolysis can induce the elevation of the TNF alpha level and pleocytosis in CSF, and whether dexamethasone can suppress this elevation of TNF alpha. We also tried to assess the effective administation timing of dexamethasone. Meningitis was induced by intracistemal inoculation of Streptococcus pneumoniae. We designed four groups : Group 1. Untreated conrol (N=5); Group 2. Lone ceftriaxone at 6 hours after inoculation (N=5) ; Group 3. Dexamethasone at 30 minutes before ceftriaxone treatment (N=5); Group 4. Dexamethasone at 30 min. after ceftriaxone treatment (N=5). CSF TNF alpha level and cell count was assessed with regular time interval. The results were as follows: First, the CSF bacterial counts (measured by colony forming units) of group 1 was significantly higher than the other groups after ceftriaxone treatment (P<0. 05). Second, the CSF WBC counts of group 3 and 4 were significantly lower than those of group 1 and 2 at 6 and 14 hours after ceftriaxone treatment (P<0.05). Third, the CSF TNF alpha titers of group 1 and 3 were significantly lower than those of group 2 and 4 at 2 hours after ceftriaxone treatment (P<0.05). Fourth, after 14 hours, the CSF TNF alpha titers of group 1 kept on rising and became significantly higher than those of other groups (P

Fenómeno de Weisser-Wechsberg (inhibición de la bacteriólisis): II. Inmunoglobulinas (IgG) de recién nacidos compiten con anticuerpos bactericidas del suero humano inmune a nivel del núcleo del lipopolisacárido (LPS-f5) de Brucella melitensis / Weisser-Wachsberg phenomenon (bactericidal inhibition): II. Newborn's immunoglobulins (IgG) compete with human immune serum's bactericide antibodies to level of lipopolysaccharide nucleus (LPS-f5) of Brucella melitensis

Introducción. El modelo de Brucellas melitensis lisa patólogena muestra que las inmunoglobulinas (IgG) de recién nacidos producen una inhibición de la bacteriólisis. No se sabe cual es la molécula implicada. Material y métodos. Por medio de liposomas se insertó lipopolisacárido (LPS) purificado de B. melitensis lisa patógena fracción 5 (LPS-f5). Se utilizó inmunotrasferencia en papel de nitrocelulosa para identificación del LPS-f5 por anticuerpos naturales e inmunes. Una prueba de bacteriólisis en cepas de B. melitensis M15 rugosa exenta de cadena lateral sirvió como modelo de inhibición al incubarse con IgG de recién nacidos y suero inmune. Resultados. Las IgG de recién nacidos remedan la bacteriólisis en un modelo liposomal. En la inmunotransferencia en papel de nitrocelulosa reconocen el núcleo de la molécula del LPS-f5 y producen inhibición de la bacteriólisis en la bacteria exenta de cadena lateral del PLS. Conclusiones. El núcleo del LPS en B. melitensis está implicado en el fenómeno de inhibición de la bacteriólisis por anticuerpos naturales

Lytic effect of Bacillus subtilis elastase on gram-positive and negative bacteria.

Elastase of B. subtilis 6a caused lysis of freshly grown cells of Gram-negative (Proteus vulgaris, Klebsiella pneumoniae, Salmonella typhi and Pseudomonas aeruginosa and Gram-positive (B. subtilis) bacteria. Heat killed and lyophilised Gram-positive and negative bacteria showed higher sensitivity to elastase. Both Gram-negative and Gram-positive bacteria were lysed maximally by elastase at pH 8.0. At this pH, activity of elastase was maximum in Tris-HCl and glycine-NaOH buffers followed by Tris-maleate and cacodylate buffers.

Capacidad fagocítica y lítica de neutrófilos en artritis reumatoidea y lupus eritematoso sistémico / Phagocytic capacity and lysis of neutrophil in rheumatoid arthritis and systemic lupus erythematosus

La capacidad fagocítica y lítica de Polimorfonucleares neutrófilos (PMNn) frente a Cándida albicans, fue evaluada en pacientes con Artritis Reumatoidea (AR) y pacientes con Lupus Eritematoso Sistémico (LES) a través de um método citomorfológico. En ambos grupos hubo una tendencia a la disminución de la función fagocítica, cuando se opsonizaron las cándidas con suero de sujetos normales, mientras que la actividad lítica evidenció un incremento solo en pacientes con AR. Hubo una reducción de la capacidad opsónica del suero de pacientes, que indujo una disminución en la función fagocítica, tanto en PMNn de pacientes como en PMNn de sujetos normales, pero con un incremento en la actividad lítica. Estos resultados sugieren que la alteración funcional observada en PMNn de estos pacientes, podría resultar de una combinación de defectos celulares y efectos de factores humorales, aunque el principal protagonista de la disfunción celular parece ser debida a componentes humorales

Bacteriolysis and variation on the O-side chain lengths of lipopolysaccharides of salmonella typhi Ty21a with respective to the concentrations of galactose

No abstract available.

Infección intrahospitalaria a Pseudomonas diminuta susceptible a piocinas / Intrahospitalary infection to pseudomanas diminuta susceptible to piocins

La incidencia de las infecciones por Pseudomonas ha incrementado especialmente en pacientes hospitalizados. En nuestro medio se identifican con cierta frecuencia Pseudomonas de diferentes materiales biológicos (orina, secreción óptica, lesiones fistulares, septicemias, catéteres y otros). Se aisló un total de 31 cepas de Pseudomonas de las cuales el 90% correspondió a P. aeruginosa y en dos casos se identificó P. diminuta, proveniente de dos pacientes internados en el hospital durante un tiempo prolongado. En uno de ellos se aisló de hemocultivo seriado y en el otro de lesiones fistulares. Ambas cepas presentaron el mismo biotipo, espectro de sensibilidad a antimicrobianos e igual susceptibilidad en la investigación de efecto inhibitorio sobre cepas del género. Este ensayo de detección de actividad bacteriocínica permitió confirmar la sospecha de una infección adquirida en el hospital.

Técnicas para evaluar la capacidad fagocítica, opsonica y bactericida del leucocito polimorfonuclear / Techniques to evaluate the phagocytic, opsonizing and bactericidal ability f the polymorphonuclear leukocyte

Para estudiar la función in vitro del leucocito PMN se estandarizaron las técnicas inmunológicas de capacidad fagocítica, opsónica y bactericida. La capacidad fagocítica se midió en sangre total, incubando leucocitos del sujeto en estudios en presencia de su propio plasma con estafilococos aureus muertos y la actividad opsónica se determinó incubando células de un individuo sano con plasma del sujeto en estudio. Los frotis teñidos se leyeron al microscopio de luz, y los resultados se expresaron como índice fagocítico, esto es número de bacterias ingeridas por 100 PMN. La capacidad bactericida se estudió, incubando una suspensión de leucocitos con bacterias (E. coli) en rotación continua a 37-C, basada en la técnica de Quie y cols. Se tomaron alícuotas para recuento de colonias por dilución en placa a los tiempos 0,20, 60 y 120 minutos. Los resultados se expresaron como índice bactericida, esto es, número de bacterias vivas a los 120 minutos/número de bacterias vivas a los 20 min. Para optimizar las técnicas se analizaron los siguientes parámetros: número de leucocitos/ml de sangre o suspensión, concentración y cepa de referencia de E. coli/ml de suspensión, período de incubación, reproducibilidad de la técnica, variabilidad intraindividual e interindividual y en el tiempo. Una vez estandarizados dichos parámetros, los índices de capacidad fagocítica y opsónica fueron de 9.2 ñ 0.85 y 8.1 ñ 0.6 (x ñ ESM), respectivamente. El índice promedio de la actividad bactericida fue de 0.14 ñ 0.05. Las pruebas fueron discriminatorias en pacientes con sospecha clínica de alteración de la capacidad funcional del leucocito