荧光法快速检测活菌数的方法研究及应用

【背景】Calcein UltraGreen~(TM)AM是一种新型荧光染料,用于标记和监测活细胞。【目的】基于该荧光染料的荧光特性及其在活细胞内的稳定特性,建立一种荧光定量快速检测活细菌总数的方法,并在实际样品中应用校正。【方法】通过应用荧光染料对细菌进行染色,再进行荧光强度检测,同时以平板计数法作平行对照,建立荧光强度值-活菌数标准曲线。【结果】确定了染色细菌的最佳pH值为8.0。该检测方法仅需固定染色温度,染色时间在20-30min范围即可快速检测。建立了革兰氏阴性菌铜绿假单胞菌NY3、大肠杆菌和革兰氏阳性菌芽孢杆菌、红平红球菌FF、金黄色葡萄球菌和枯草芽孢杆菌的细菌总数与相对荧光强度值标准曲线。当菌悬液OD600值在0.01-0.30范围内时,上述6种细菌与荧光信号强度呈良好的线性关系(R20.99)。【结论】当样品菌悬液浓度范围控制在105-109CFU/mL时,建立的荧光检测方法快速便捷,精密度、重复性、稳定性、回收率和准确度均较好,可应用于微生物实验、固体菌剂发酵、食品卫生与安全、环境检测等领域的活细菌总数现场快速检测。     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Variations in concentrations of bacterial metabolites, enzyme activities, moisture, pH and bacterial composition between and within individuals in faeces of seven healthy adults

Variations between and within individuals, and correlations between concentrations of bacterial metabolites, including putrefactive products, ammonia and short chain fatty acids (SCFAs), enzyme activities, moisture and pH, as well as bacterial composition, were studied in faecal samples from seven healthy adults over a period of 7 months. Large variations, both between and within individuals, were observed in concentrations of putrefactive products. Although values for ammonia, SCFAs, enzyme activities, moisture and pH were generally variable, significant person-to-person differences were observed.
While ranges of log viable counts of the predominant bacteria such as eubacteria, bifidobacteria and bacteroides in each subject remained between 0·2 and 1·3, those of enterobacteria, streptococci (including enterococci) and lecithinase-negative clostridia varied between 0·4 and 3·0. Levels of bifidobacteria, enterobacteria, streptococci and total aerobic bacteria showed inter-individual variations. Correlations were found among certain of the parameters: moisture correlated negatively with p -cresol ( r = -0·707), pH ( r = -0–671) and β-glucosidase activity (GS) ( r = -0·608), and positively with acetic acid ( r = 0·621), while negative correlations were observed in pH with acetic and butyric acids ( r = -0·690 and -0·623, respectively).
No significant correlations were found between bacterial compositions, and other faecal factors such as pH, moisture, metabolic enzyme activities and concentrations of putrefactive products.     

Differential effect of prior influenza infection on alveolar macrophage phagocytosis of Staphylococcus aureus and Escherichia coli: involvement of interferon-gamma production

The influenza A virus is one of the main causes of respiratory infection. Although influenza virus infection alone can result in pneumonia, secondary bacterial infection combined with the virus is the major cause of morbidity and mortality. Interestingly, while influenza infection increases susceptibility to some bacteria, including Streptococcus pneumoniae, Staphylococcus aureus (S. aureus), and Haemophilus influenzae, other bacteria such as Escherichia coli (E. coli) and Klebsiella pneumoniae are not associated with influenza infection. The reason for this discrepancy is not known. In this study, it was found that prior influenza virus infection inhibits murine alveolar macrophage phagocytosis of S. aureus but not of E. coli. Here, the mechanism for this inhibition is elucidated: prior influenza virus infection strongly increases interferon gamma (IFN-γ) production. Furthermore, it was shown that IFN-γ differentially affects alveolar macrophage phagocytosis of S. aureus and E. coli. The findings of the present study explain how influenza virus infection increases susceptibility to some bacteria, such as S. aureus, but not others, and provides evidence that IFN-γ might be a promising target for protecting the human population from secondary bacterial infection by influenza.     

Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli

The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log(10) CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.     

A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157 : H7 in beef using direct epifluorescent microscopy and immunomagnetic separation

An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157 : H7 cells (0·15 cells g⁻¹) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42°C in an orbital shaking water bath (200 rev min⁻¹). Over 50% of the microscopic fields viewed were positive (1–10 fluorescent cells field⁻¹) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads^® anti- E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-β- D -glucuronide. At this cell concentration, 41·7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0·58% formaldehyde concentration for 5 min at 50°C (killed >4·00 logs of E. coli O157 : H7 cells) without affecting cell fluorescence.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

The use of Alcalase 2·5L in the acridine orange direct count technique for the rapid enumeration of bacteria in beef mince

The acridine orange direct count (AODC) technique was used to count bacterial numbers in beef mince. The ability of Alcalase 2·5L to degrade beef mince was compared with the previously used Alcalase 0·6L. Both methods were evaluated against the standard plate count. The results showed that Alcalase 2·5L can be used in the AODC technique for the rapid counting of bacterial numbers in beef mince. Prediction equations obtained for the relationship between the AODC and standard plate counts were validated with commercial beef mince samples.     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims:  The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water.
Methods and Results:  Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r  = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci.
Conclusions:  The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria.
Significance and Impact of the Study:  The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.     

A rapid method for determining the quality of gari based on iodine reduction test

A rapid iodine reduction method was developed for predicting the quality of gari. Good quality samples took longer (21·0 ± 4·5s) to reduce iodine than poor quality samples (2·2 ± 0·42 s). There was a strong association between quality and reduction time ( r = 0·97, df = 12) and storage period ( r = 0·99, df = 12) and the reduction time was also dependent on bacterial numbers. Gari stored at low temperatures contained fewer micro-organisms and retained its quality longer than that kept at higher temperatures. The quality of gari can be predicted from iodine reduction times.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

Fluoro-luminometric real-time measurement of bacterial viability and killing

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.     

The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin–EDTA combination and their effectiveness as antibiofilm agents

Aims:  To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa , Escherichia coli and Candida albicans in planktonic and biofilm cultures.
Methods and Results:  Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1·2 to 2·7 mg ml⁻¹. Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1·35 and 3·83 mg ml⁻¹. A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination.
Conclusion:  Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa , E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication.
Significance and Impact of the Study:  There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.     

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action.

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.     

Changes in microbial population during fermentation of tropical freshwater fish viscera

Freshwater fish viscera (FV) was homogenized, mixed with 10% (w/w of FV) molasses and 0, 2 or 4% salt and allowed to ferment at ambient temperature (26·2°C) under microaerophilic conditions. The results revealed a reduction in total viable count and the number of spores, coliforms, Escherichia coli , staphylococci and enterococci and an increase in yeasts and moulds and lactic acid bacteria during fermentation. Coliforms and E. coli were found to be absent after 6 d and enterococci on 8th day. The presence of salt resulted in a marginally lower number of all organisms except yeasts, moulds and lactic acid bacteria. Inclusion of either 0·5% propionic acid, 0·3% calcium propionate or 0·1% sorbic acid suppressed growth of yeasts and moulds with propionic acid being the most effective. The study indicated that a microbiologically stable product could be prepared by ensiling fish viscera with 10% molasses and 0·5% propionic acid.