Modified Microbiological Assay for Rapid Estimation of Antibiotic Concentrations in Human Sera

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.     

Lysis by β-lactam Antibiotics: Structure-activity Relationships in the Cephalosporins

The ability of several cephalosporins to lyse Staphylococcus aureus and Escherichia coli has been studied. Cephradine and cephalexin, which have a methyl substituent at C-3, did not lyse E. coli , but a third 3-methyl compound, deacetoxycephalothin, caused bacteriolysis. All the compounds tested (including cephalexin and cephradine) lysed Staph. aureus . The minimum lytic concentration of a cephalosporin for E. coli often exceeded its minimum inhibitory concentration, while for staphylococci this was never the case.     

Specific Antimicrobial Synergism of Synthetic Hydroxy Isothiocyanates with Aminoglycoside Antibiotics

Hydroxy isothiocyanates, especially 2-(4-hydroxy-phenyl)ethyl isothiocyanate (hITC), were examined for antimicrobial synergism with several antibiotics against Escherichia coli and Staphylococcus aureus, using a multiwell plate system. hITC had antibacterial synergism, specifically with aminoglycoside antibiotics. The synergism was observed in synthetic medium (M9 minimal medium) or soybean casein digest broth, but not in nutrient broth. Synergism was seen in the presence of certain sugars such as glucose, fructose, and maltose in the medium.     

Strain-Specific Neutralization of Human Cytomegalovirus Isolates by Human Sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

检测肾综合征出血热抗原特异性循环免疫复合物ELISA方法的建立

检测肾综合征出血热抗原特异性循环免疫复合物ＥＬＩＳＡ方法的建立张东海,孙辉,高峰（山东省淄博第二卫生学校,淄博２５５０１５）关键词肾综合征出血热病毒,循坏免疫复合物,特异性,ＥＬＩＳＡ肾综合征出血热（ＨＦＲＳ）发病机理有多种学说,其中在体液免疫学说中...     

Unsaturated Fatty Acid,cis-2-Decenoic Acid,in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms'' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.     

Production and Reactivity of Immune Sera Specific for HADEN Virus Polypeptide Antigens

Antisera were prepared against the three structural polypeptides of HADEN virus dissociated with sodium dodecyl sulfate. These immunological reagents were used in immunofluorescence tests to study the kinetics and location of polypeptide antigen appearance in infected cells. These sera did not neutralize virus infectivity, did not cross-react with adenovirus-associated virus-infected cells, and reacted in complement fixation tests with sodium dodecyl sulfate-dissociated virus, but not with complete virus antigen. The polypeptide antigens were heat labile, and all appeared in infected cells at least 2 h prior to whole-virus antigen.     

E.tenella感染雏鸡特异性抗体的动态变化和母源免疫的研究

本实验用斑点酶联免疫试验 (Dot ELISA)检测了E .tenella感染雏鸡特异性抗体、免疫母鸡卵黄和后代血清中特异性母源抗体的动态变化及不同日龄雏鸡的抗球虫水平。结果表明 :(1)雏鸡感染E .tenella后第 6d即可在循环血液中检测到特异性IgG ,于第 18天达到峰值 ,第 30天降至感染后第 7天时的水平。 (2 )母鸡二次免疫后第 5天卵黄中即有高水平的母源抗体 ,免疫母鸡后代 1日龄时母源抗体滴度高达 9.83,与未免疫对照组差异极显著 (P <0 .0 1) ,二者都随着日龄增长降低 ,但免疫组下降幅度很小 ,2 9日龄时仍和对照组 18日龄水平相当。 (3)免疫母鸡后代对再感染的抵抗力显著增强 ,卵囊减少率高达 93%,抗球虫指数和相对增重率都与对照组差异极显著 (P <0 .0 1) ,雏鸡的抗球虫水平随着日龄增长逐渐降低 ;母源抗体、抗球虫指数和相对增重率三者之间两两呈现明显的正相关 ,而这三者又都与卵囊产量呈明显的负相关。     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

Radiosterilization of Fluoroquinolones and Cephalosporins: Assessment of Radiation Damage on Antibiotics by Changes in Optical Property and Colorimetric Parameters

A most common problem encountered in radiosterilization of solid drugs is discoloration or yellowing. By pharmacopoeia method, discoloration can be assessed by measuring absorbance of solutions of irradiated solid samples at 450 nm. We propose to evaluate discoloration of solid samples directly by recording their diffuse reflectance spectra. Further, the reflectance spectrum is used to compute various color parameters: CIE XYZ tristimulus value, CIE Lab, (color difference), yellowness index (YI), dominant wavelength, and excitation purity by CIE method. The investigation of difference reflectance spectra and color parameters revealed that for fluoroquinolones, e-beam was more damaging than gamma radiation, whereas for cephalosporins, trend was reversed. The quantum of discoloration with gamma radiation and e-beam is found to be nearly equal when assessed by pharmacopeia method, and it is therefore inadequate to assess small color differences. The color parameters and ΔYI are found to be reliable indicators of discoloration. The tolerance limits proposed in terms of and ΔYI are ±2 and ±10 U, respectively. The dominant wavelength for all compounds has shifted to higher values indicating change in hue but defining color tolerance limit with this parameter requires adjunct excitation purity value.     

Extremely High Frequency Electromagnetic Irradiation in Combination with Antibiotics Enhances Antibacterial Effects on Escherichia coli

Antibacterial effects of the electromagnetic irradiation (EMI) of 51.8 and 53 GHz frequencies with low intensity (the flux capacity of 0.06 mW/cm(2)) and non-thermal action were investigated upon direct irradiation of E. coli K12. Significant decrease in bacterial growth rate and in the number of viable cells, marked change in H(+) and K(+) transport across membrane were shown. Subsequent addition of kanamycin or ceftriaxone (15 or 0.4 μM, respectively) enhanced the effects of irradiation. This was maximally achieved at the frequency of 53 GHz. These all might reveal membrane as probable target for antibacterial effects. Apparently, the action of EMI on bacteria might lead to changed membrane properties and to antibiotic resistance. The results should improve using extremely high frequency EMI in combination with antibiotics in biotechnology, therapeutic practice, and food industry.     

Physical Assay of Competence for Specific Mating-Pair Formation in Escherichia coli

The fractions of Escherichia coli female and male bacteria that are competent to form specific mating pairs at any time during normal exponential growth have been measured by a physical assay. The competencies of a Hayes- and a Cavali-type donor were 35 and 50%, respectively, in agreement with piliation assays reported by others. The competencies of two recipient strains, PA309 and P678, were found to be 33 and 29%, respectively.     

Prevention of Staphylococcal Bacteriophage Activity by Antigen A Precipitins in Human Sera

Antigen A precipitins in human sera prevented plaque formation and propagation of staphylococcal bacteriophages. Over 20% of total IgG was removed from human sera by absorption with staphylococci containing antigen A. The specific precipitating antibody in rabbit antisera formed lines of idenity with antigen A precipitins in lower dilutions of human sera but formed lines of nonidenity with antigen A precipitins in higher dilutions of the same sera, suggesting both specific and nonspecific antigen A precipitins in human sera. The specific and nonspecific antigen A precipitins in human sera may prevent the in vivo activity of staphylococcal bacteriophages which have been demonstrated previously in animals whose sera do not contain either specific or nonspecific antigen A precipitins.     

Occurrence of Antibodies to Group Specific Chlamydia Antigen in Cattle and Reindeer Sera in Finnish Lapland

The occurrence of group specific complement fixing antibodies was studied in random sera of cattle and reindeer in Finnish Lapland. Sixty-eight (40.5%) of the 168 cattle sera were positive. Sixty 4hree (21.6 %) of the 291 reindeer sera were positive. The difference is statistically nearly significant in the t-test. The antibody titer ≥ 1:16 was regarded as positive. The antibody frequency of cattle sera was statistically significantly higher and the antibody frequency of reindeer sera was nearly significantly higher than in earlier studies on cattle sera in South and Central Finland. The reasons are discussed.