Determination of beta-galactosidase activity on nitrocellulose plates using 5-bromo-3-indolyl-beta-D-galactopyranoside and tetrazolium salts

A simple and convenient technique has been developed for detection of beta-galactosidase from E. coli on nitrocellulose sheets using a mixture of 5-bromoindol-3-yl-beta-D-galactopyranoside and nitro blue tetrazolium, which enables rapid detection of fmole (10(-15) mole) quantities of the enzyme at pH 9.5. The technique has the following advantages: the substrates are stable for a long period; reaction products give non-fading intense blue colour, resolution is extremely good with essentially no diffusion.     

Pollution indicator bacteria associated with municipal raw and drinking water supplies.

A total of 3819 bacterial cultures isolated from municipal water samples were identified using a combination of Enterotubules and confirmatory media. Frequency distributions for the different genera or groups of bacteria were similar for raw water and drinking water isolations, except for Escherichia organisms which doubled their frequency in raw water. Differences between the membrane filter (MF) and presence-absence (P-A) test with regard to types of organisms isolated were limited to Klebsiella organisms which were preferentially cultured from MF plates. Members of the genus Enterobacter were isolated more than twice as frequently as any of the other coliform genera dealt with in this study. Aeromonas organisms were detected almost as often as such individual genera as Escherichia, Citrobacter, or Klebsiella. Although non-lactose fermenting colonies (false-negatives) of the coliform genera would not be detected by the MF technique, their lack of detection would likely be offset by the Aeromonas colonies (false-positives). At least 25% of the coliform isolates were either anaerogenic or non-lactose fermenters and would therefore go undetected by the most probably number (MPN) technique.     

利用X-Neu5Ac为底物测定唾液酸酶活性在诊断细菌性阴道病的价值

目的利用5溴-4氯-3吲哚乙酰基神经氨酸盐（X-Neu5Ac）为底物测定阴道唾液酸酶活性诊断细菌性阴道病（bacterial vaginosis,BV）的价值.方法健康妇女30例,临床Amsel法诊断为BV的患者45例,共计75例进行了阴道分泌物分析和检测,并与唾液酸酶活性法诊断作了对比研究.取阴道分泌物作为标本分别进行唾液酸酶活性和阴道菌群定量分析,检测细菌包括乳酸杆菌、类杆菌、肠杆菌、葡萄球菌、肠球菌和阴道加德纳菌.唾液酸酶活性测定利用的底物为X-Neu5Ac,特异活性用其产物 ——甲氧基苯酚的纳摩尔数来表示.结果阴道液唾液酸酶活性测定诊断细菌性阴道病的敏感性、特异性、阳性预期值和阴性预期值分别为88.9%、90%、93%和84.3%.唾液酸酶法在检测细菌性阴道病上和传统的Amsel法比较,差异无显著性（P＞0.05）.唾液酸酶阳性组Gv活菌数（6.96 log CFU/g）明显高于唾液酸酶阴性组（2.05 log CFU/g）（P＜0.01）.唾液酸酶阳性组产H2O2阴道乳杆菌（LB＋）活菌数（4.26 Log CFU/g）明显低于唾液酸酶阴性组（8.66 Log CFU/g）（P＜0.01）.唾液酸酶阳性组与唾液酸酶阴性组两组的阴道液中需氧菌活菌数差异无显著性（P＞0.05）,主要包括金黄色葡萄球菌、肠球菌和肠杆菌.结论利用X-Neu5Ac作为唾液酸酶的底物测定唾液酸酶活性的方法是诊断细菌性阴道病的有效检测方法.阴道内唾液酸酶活性增强,厌氧菌数量增加,LB＋数量减少,提示BV发生恶化.     

The effect of bisulfite on the dehalogenation of 5-chloro-, 5-bromo-, and 5-iodouracil

The 5-chloro-, bromo-, and iodo-analogs of uracil are dehalogenated in the presence of sodium bisulfite to yield 5,6 dihydrouracil-6-sulfonate as the final product. Under similar conditions, 5-fluorouracil adds bisulfite to yield 5-fluoro-5,6 dihydrouracil-6-sulfonate but is not dehalogenated. Ultraviolet absorption spectra of 5-bromouracil and 5-iodouracil reacting under pseudo first-order conditions with bisulfite indicate that dehalogenation proceeds via a pathway which has 5-halo-5,6-dihydrouracil-6-sulfonate and uracil as intermediates. In the case of 5-chlorouracil, the rate of bisulfite attack on the 6-position of the chlorouracil ring system is very slow relative to the rate of bisulfite addition to uracil. Hence, although dechlorination does occur, ultraviolet absorption spectra of reaction mixtures containing bisulfite and 5-chlorouracil do not reveal the uracil absorption peak observed with both 5-iodouracil and 5-bromouracil. Fluorine and proton nmr spectra indicate that bisulfite addition to 5-fluorouracil is stereoselective as is the case of bisulfite addition to uracil.     

Chemiluminescent enzyme immunoassay for alpha-fetoprotein using β-D-galactosidase as label and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as substrate

We developed a sensitive chemiluminescent sandwich-type enzyme immunoassay (CLEIA) of alpha-fetoprotein (AFP) using b?-D -galactosidase (b?-gal) as a label and 5-bromo-4-chloro-3-indolyl-b?-D -galactopyranoside as a substrate. The CL-EIA for AFP was performed using two monoclonal antibodies, one antibody is labelled with b?-gal, the other is coated onto the inside surface of a polystyrene tube. The detection limit for AFP was 0.5 ng/mL, equivalent to 10 pg/assay tube. The coefficient of variation for within and between assay imprecision were 2.0%?4.9% (n = 10) and 4.4%?9.8% (n = 5), respectively. AFP values in serum determined by this method correlated well with those obtained by radioimmunoassay (n = 26, r = 0.99). This sensitive AFP assay can be performed within 4 h and can be used as a routine assay in clinical diagnosis.     

Factors promoting survival of bacteria in chlorinated water supplies

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

Factors promoting survival of bacteria in chlorinated water supplies.

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from antarctic soil

Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10 degrees C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45 degrees C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: alpha-, beta-, and gamma-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.     

The use of antibodies to 5-bromo-2'-deoxyuridine for the isolation of DNA sequences containing excision-repair sites

We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.     

Syntheses of 4-methylumbelliferyl-beta-D-xylobioside and 5-bromo-3-indolyl-beta-D-xylobioside for sensitive detection of xylanase activity on agar plates

4-Methylumbelliferyl-beta-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-beta-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.     

The in vivo construction of 4-chloro-2-nitrophenol assimilatory bacteria

Pseudomonas sp. N31 was isolated from soil using 3-nitrophenol and succinate as sole source of nitrogen and carbon respectively. The strain expresses a nitrophenol oxygenase and can use either 2-nitrophenol or 4-chloro-2-nitrophenol as a source of nitrogen, eliminating nitrite, and accumulating catechol and 4-chlorocatechol, respectively. The catechols were not degraded further. Strains which are able to utilize 4-chloro-2-nitrophenol as a sole source of carbon and nitrogen were constructed by transfer of the haloaromatic degrading sequences from either Pseudomonas sp. B13 or Alcaligenes eutrophus JMP134 (pJP4) to strain N31. Transconjugant strains constructed using JMP134 as the donor strain grew on 3-chlorobenzoate but not on 2,4-dichlorophenoxyacetate. This was due to the non-induction of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase. Transfer of the plasmid from the 2,4-dichlorophenoxyacetate negative transconjugant strains to a cured strain of JMP134 resulted in strains which also had the same phenotype. This indicates that a mutation has occurred in pJP4 to prevent the expression of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase.     

Use of methylotrophic bacteria Methylobacillus flagellatum KT for isolation of deuterated exogenous carbohydrates

The cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, while deuterated exogenous polysaccharides included 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Efficient bioreduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydrobutanoate by whole cells ofCandida magnoliae in water/n-butyl acetate two-phase system

The asymmetric biosynthesis of ethyl (S)-4-chloro-3-hydrobutanoate from ethyl 4-chloro-3-oxobutanoate was investigated by using whole cells ofCandida magnoliae JX120-3 without the addition of glucose dehydrogenase or NADP⁺/NADPH. In a one-phase system, the bioconversion yield was seriously affected on the addition of 12.1 g/L ethyl 4-chloro-3-oxobutanoate. In order to reduce this substrate inhibition, a water/n-butyl acetate two-phase system was developed, and the bioreduction conditions optimized with regard to the yield and product enantiometric excess value. The optimal conditions were as following: water ton-butyl acetate volume ratio of 1∶1, 4.0 g DCW/L active cells, 50 g/L glucose and 35°C. By adopting a dropwise substrate feeding strategy, high concentration of ethyl 4-chloro-3-oxobutanoate (60 g/L) could be asymmetrically reduced to ethyl (S)-4-chloro-3-hydrobutanoate with high yield (93.8%) and high enantiometric excess value (92.7%).     

Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics

Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.     

Strategies for isolation of in vivo expressed genes from bacteria

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of the infection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encode virulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification of these genes is expected to provide new means to prevent infection, new targets for, antimicrobial therapy, as well as new insights into the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed by functional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makes feasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-tagged mutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterial genomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterial pathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo.