A shortcut method for estimation of Escherichia coli in Sewage and receiving water

A simplified method not yet widespread in Scandinavia for the assessment of E. coli in sewage and recipients with McConkey broth for 24 hours at 44 degrees C is recommended. 87 pairs of samples were examined at 37 degrees C and later at 44 degrees C, and directly at 44 degrees C. Also the reduction of the incubation time from 48 to 24 hours was studied. Direct incubation gave a loss of half a tube per sample, while shortening of the incubation period from 48 to 24 hours gave a smaller loss. The proposed alteration of the procedure for the examination of sewage and recipients means a slight loss of precision but this is more than compensated for by the possibility of examining more samples. The survey of the recipients can therefore be better and the control more effective.     

Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water

Two media used to detect fecal coliforms in water by membrane filtration, m-FC and m-TEC, were modified and supplemented with the chromogenic substrate 5-bromo-6-chloro-3-indoyl-beta-D-glucuronide (BCIG) and were compared for quantitative recovery of Escherichia coli. Student's t test of data from 181 water samples of sewage, rivers, lakes, and wells did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli with these media. Target colonies were confirmed to be E. coli at rates of 98.6 and 97.3% by using FC-BCIG and TEC-BCIG media, respectively. Glucuronidase-negative isolates of E. coli were encountered at the same frequency (6.0%) on both media. This collaborative study demonstrated that either modified basal medium could be used successfully for detection of E. coli in various nontreated waters within 24 h.     

Antimicrobial action of rabbit leukocyte CAP18(106-137)

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.     

Comparison of two enrichment methods and five selective media for the isolation of Yersinia enterocolitica from tonsils of slaughter pigs (author's transl)

115 tonsils of healthy slaughter pigs were culturally examined for presence of Yersinia (Y.) enterocolitica. For this purpose each sample was enriched both in phosphate buffered saline solution (pH 7.6; stored at 4 degrees C and plated every week, thrice all together) and modified Rappaport broth (plated after an incubation of two days at 22 degrees C). Each such enrichment was plated on 5 different selective media: Yersinia selective agar proposed by Wauters (1973), deoxycholate-citrate-mannitol agar (Saari and Jansen, 1979), pectin agar (Bowen and Kominos), MacConkey and Leifson agar as used in the routine, diagnostic of Enterobacteriaceae. Each agar plate was incubated at 28 degrees C for two days. By cold enrichment method were isolated 11 strains of human pathogenic Y. enterocolitica (9 X O-group I syn. serotype O:3; 2X O-group V syn. serotype O:9). With the modified Rappaport medium were recovered 33 strains (24X O-group I, 9 X O O-group V). The most recoveries were done over the Yersinia selective agar with 65.2%, then followed deoxycholate-citrate-mannitol agar with 57.6%, Leifson agar with 45.5%, MacConkey agar with 42.3% and pectin agar only with 18.2% of the isolations. Not only the type of enrichment medium has a marked effect in the recovery efficiency of Y. enterocolitica out of samples but also number and type of the used selective media on which the enrichment is plated.     

Use of total of Escherichia coli counts to assess the hygienic characteristics of a beef carcass dressing process

Swab samples were obtained from 3 sites on the surfaces of beef carcasses passing through a high speed dressing process, with 24 samples from each site being obtained at each of 4 points in the process. The aerobic microflora recovered from each swab after incubation at 25 degrees C was enumerated and characterized, and numbers of coliforms and Escherichia coli were determined. The data on aerobic flora indicated that skinning results in similar contamination of all 3 sites, that further deposition of bacteria at the brisket site occurs after skinning, and that trimming and washing achieve modest decontamination of the neck and brisket site, and extensive decontamination of the rump site. Changes in flora compositions during processing were too limited to much affect the assessment based on the aerobic flora total counts alone. The E. coli data indicated that during skinning the rump site was more heavily contaminated with faecal organisms than the other sites, that contamination of the brisket site is little altered between skinning and carcass splitting, although there is an extensive redistribution of E. coli at the neck site and sporadic, limited decontamination of the rump site, and that trimming and washing do not decontaminate the neck or rump sites, but that the rump site is extensively decontaminated by trimming. There was good correlation between E. coli and coliform counts, but weak correlation between E. coli and aerobic, 25 degrees C, counts. The findings suggest that assessments of beef carcass dressing processes for Hazard Analysis: Critical Control Point (HACCP) purposes should be based on enumerations of E. coli, or perhaps coliforms, rather than of the aerobic flora, to avoid important misunderstandings of the hygienic effects of the various operations in a process.     

Posttransplant lymphoproliferative disorder in pediatric bone marrow transplant recipients: disseminated disease of donor origin demonstrated by fluorescence in situ hybridization

Two new antibiotics, hongoquercins A and B, were isolated from fermentation extracts of the unidentified fungus LL-23G227. In the optimum medium, titers of the A and B components reached approximately 2.1 g/liter and 0.02 g/liter, respectively. The optimum temperature for antibiotic production was approximately 22 degrees C. Growth was delayed at 15 degrees C but appeared to reach higher levels than was observed at 22 degrees C. Addition of dextrose to growth media increased hongoquercin B production. Hongoquercin A exhibited moderate activity against Gram-positive bacteria. Mechanistic studies conducted in an E. coli imp strain suggested membrane damage as the primary mode of bactericidal action. These compounds also lysed human red blood cells, suggesting a similar mode of action on eukaryotic cells.     

Evaluation of a new disk containing indoxyl-beta-D-glucuronide for rapid identification of Escherichia coli

To establish a simple identification procedure for Escherichia coli, we developed a disk containing indoxyl-beta-D-glucuronide, the chromogenic substrate of beta-D-glucuronidase. Of 188 isolates of Enterobacteriaceae, 101 (97%) of 104 strains of E. coli and two (67%) of three strains of Shigella species were positive for beta-glucuronidase. We also tested 495 strains (466 strains of E. coli and 29 of Citrobacter freundii) that might be considered lactose-fermenting E. coli under routine conditions, and 458 strains of E. coli showed beta-glucuronidase activity: the sensitivity of the disk method was 92%, specificity was 100%, and the negative predictive value was 44%. Our results suggest that for routine identification of E. coli on primary isolation plates, the disk method is sufficiently rapid (within 3h), simpler, and far less costly than identification methods using commercially available kits.     

Comparison of Rambach agar, SM-ID medium, and Hektoen Enteric agar for primary isolation of non-typhi salmonellae from stool samples

Stool samples (n = 504) were streaked simultaneously onto Rambach agar (R agar; E. Merck, Darmstadt, Germany), SM-ID medium (bioMérieux S.A., Montalieu-Vercieu, France), and Hektoen Enteric (HE) agar (BBL Becton-Dickinson, Baltimore, Md.) in order to evaluate the performances of the first two media in comparison with that of the well-established HE agar. Following overnight cultivation at 37 degrees C, 29 samples (5.8%) were positive for non-typhi salmonellae on at least one of the three media. Sensitivities and specificities were 69 and 98%, 79 and 85%, and 100 and 79% for R, SM-ID, and HE agars, respectively. On the basis of the poor sensitivities, R and SM-ID agars are not recommended as primary plating media when screening for non-typhi salmonellae. However, the high specificity of R agar may help to reduce the work load when this medium is used for plating after enrichment.     

Temperature determines the pattern of anaerobic microbial dechlorination of Aroclor 1260 primed by 2,3,4,6-tetrachlorobiphenyl in Woods Pond sediment

Reductive dechlorination of the Aroclor 1260 residue in Woods Pond (Lenox, Mass.) sediment samples was investigated for a year at incubation temperatures from 4 to 66 degrees C. Sediment slurries were incubated anaerobically with and without 2,3,4,6-tetrachlorobiphenyl (2346-CB; 350 microM) as a primer for dechlorination of the Aroclor 1260 residue. Dechlorination of the Aroclor residue occurred only in live samples primed with 2346-CB and only at 8 to 34 degrees C and 50 to 60 degrees C. The extent and pattern of polychlorinated biphenyl (PCB) dechlorination were temperature dependent. At 8 to 34 degrees C, the dechlorination resulted in 28 to 65% decreases of the hexathrough nonachlorobiphenyls and corresponding increases in the tri- and tetrachlorobiphenyls. At 12 to 30 degrees C, 30 to 40% of the hexa- through nonachlorobiphenyls were dechlorinated in just 3 months. The optimal temperature for overall chlorine removal was 20 to 27 degrees C. We observed four different microbial dechlorination processes with different but partially overlapping temperature ranges, i.e., Process N (flanked meta dechlorination) at 8 to 30 degrees C, Process P (flanked para dechlorination) at 12 to 34 degrees C, Process LP (unflanked para dechlorination) at 18 to 30 degrees C, and Process T (a very restricted meta dechlorination of specific hepta- and octachlorobiphenyls) at 50 to 60 degrees C. These temperature ranges should aid in the development of strategies for the enrichment and isolation of the microorganisms responsible for each dechlorination process. The incubation temperature determined the relative dominance of the four PCB dechlorination processes and the extent and products of dechlorination. Hence, understanding the effects of temperature on PCB dechlorination at contaminated sites should assist in predicting the environmental fate of PCBs or planning bioremediation strategies at those sites.     

Microbial inhibitory properties and stability of topotecan hydrochloride injection

The viability of five microorganisms in topotecan 1 mg/mL (as the hydrochloride salt) in sterile water and the stability of the drug were studied. Duplicate portions of topotecan 1 mg/mL were inoculated with Escherichia coli. The process was repeated for Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus niger. Samples were removed from each solution initially and after 6, 16, and 24 hours and 3, 7, 14, 21, and 28 days of incubation at 20-25 degrees C. To test stability, vials of reconstituted topotecan hydrochloride injection were stored at each of three temperatures--5, 25, and 30 degrees C--and other vials were used for time zero analysis. For each temperature, vials were removed at 1, 7, and 14 days and the remaining vials at 28 days for analysis by high-performance liquid chromatography and for visual and pH assessment. P. aeruginosa, S. aureus, and E. coli lost viability at 16 hours, 24 hours, and 28 days, respectively. C. albicans and A. niger did not lose viability, but their numbers did not grow. No differences in color or clarity were observed, and pH was constant. In all solutions, the topotecan concentration was > 98% of the initial concentration. Topotecan 1 mg/mL in sterile water stored at 20-25 degrees C for up t 28 days did not support growth of the five microorganisms studied; in solutions stored at 5, 25, or 30 degrees C for up to 28 days, topotecan 1 mg/mL remained stable.     

Effectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures

The behavior of Escherichia coli O157:H7 inoculated in 10% rehydrated nonfat dry milk adjusted to pH levels between 3.8 and 5.4 with lactic acid, salt levels of 0 to 6%, and diacetyl levels of 0, 5, and 10 micrograms/g was determined at 4 and 12 degrees C. Cell populations were determined by surface plating on tryptic soy agar after 7 and 35 days of incubation. Survival was also determined using retail cultured diary products. E. coli O157:H7 did not survive in skim milk at pH 3.8 and was reduced by 3 log cycles at pH 4.1, regardless of salt, diacetyl, and temperature levels. At pH levels above 4.4, survival was observed at lower salt concentrations for up to 35 days at both 12 and 4 degrees C. The organism grew (up to a 2.2-log increase) at pH 5.0 at 2% salt levels after 35 days of storage at 12 degrees C. Diacetyl at a concentration of 10 ppm had no effect on survival and growth. In all but one case, E. coli O157:H7 was inactivated in yogurt, sour cream, and buttermilk at a rate similar to or greater than what was consistent with the acidified skim milk data. Also consistent with the skim milk data, growth occurred in two of the three cottage cheese samples at 12 degrees C after 7 days but not after 35 days or at 4 degrees C, when a 1- to 2-log decline was observed.     

A comparison of brain heart infusion blood agar sterilized by filtration and heat on the growth of Neisseria gonorrhoeae

The growth of Neisseria gonorrhoeae on brain heart infusion blood agar in which the base was sterilized by filtration has been compared with growth on the same medium sterilized by heat. Colonies were larger on the unheated medium, and autoclaving at 115 degrees C of 121 degrees C for 15 minutes was accompanied by a progressive decrease in colony size. Viable counts on the three media showed no difference in end points. Colonies on the unheated medium were usually large enough to be easily recognizable after overnight incubation.     

Comparison of BGB-MUG and LSTB-MUG in microbiological surveillance of recreational waters

Recreational water surveillance is an important tool to prevent health hazards for the population. Therefore distinct guide and imperative values for fecal indicators are listed in the EC directive about water quality control. The detection methods, however, give laboratories some room to choose their own method, which has led to difficulties in the comparability of results. In 1989 an ad-hoc working group of the coastal countries of Germany established detection methods, which by now are obligatory for these countries. Fecal and total coliforms (FC and TC) are detected by a triplicate mpn-procedure using brilliant green-bile-lactose broth supplemented with tryptophane and 4-methylumbelliferyl-beta-D-glucuronide (BGB-MUG) as selective medium. Gas-, fluorescence- and indole-positive cultures are considered fecal coliform-positive. In the last years rises in TC but not in FC counts were observed in fresh waters. A study was carried out to evaluate the official method in another bathing season, to determine bacterial species leading to false-positive TC cultures and to compare BGB-MUG with laurylsulphate-tryptophane-MUG (LSTB-MUG). Water samples of different salinities and nutrient input were collected in weekly intervals from April to October. FC and TC concentrations were determined and all TC-positive cultures were differentiated further. The FC counts obtained by enrichment in BGB-MUG or LSTB-MUG were nearly identical, the rate of fluorescence-positive, indole-negative tubes being approximately 0.6%. Differentiation of FC-negative cultures showed a false-negative rate of 2.87% for BGB-MUG and of 8% for LSTB-MUG. During the summer months TC counts in BGB-MUG exceeded FC counts by far at most of the sampling sites. This effect was much less pronounced in LSTB-MUG; the difference between both enrichment media being significant. Differentiation of presumptive TC from BGB-MUG resulted in a high percentage of Aeromonas spp. in fresh waters. LSTB-MUG was clearly more selective for TC than BGB-MUG, but still with an average of 10% of the test tubes being false TC-positive (BGB-MUG 46%). The sensitivity of BGB-MUG was below 60% (LSTB-MUG 89%). LSTB-MUG should be preferred as enrichment medium in mpn-examination of recreational water, if no further differentiation is carried out. The selectivity for TC is better than in BGB-MUG and the only slight inhibitory effects can be tolerated.     

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10 g of ground beef when samples were enriched in modified EC broth for 7 h at 37 degrees C. The minimum detectable number of cells for the apple cider samples was calculated to be approximately 0.5 cfu ml-1. The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.     

Studies on enrichment broth for verotoxin-producing Escherichia coli (VTEC) O157

Growth of verotoxin-producing Escherichia coli (VTEC) O157 in conventionally recommended pre-enrichment broth media at different temperatures was evaluated. In addition, sensitivity of VTEC O157 isolates to several antibacterial drugs, which were added to the selective enrichment broth, was tested. All five isolates of VTEC O157 tested grew well in trypticase soy broth (TSB) at 36 degrees C and 42 degrees C, while the growth of one isolate was markedly suppressed in TSB supplemented with cefixime (CFIX), potassium tellurite (PT), and vancomycin (TSB-CTV) even at 36 degrees C. A significant growth suppression was also observed in three of the isolates cultured in novobiocin (NB)-supplemented modified EC broth (mEC-NB) at 42 degrees C. In mEC-NB after 24-hr incubation at 36 degrees C, the five VTEC O157 isolates grew well, although one isolate was slightly suppressed during the first 8 hours. Minimum growth inhibitory concentrations of CFIX, NB and PT for a total of 90 clinical and environmental isolates of VTEC O157 were all above the concentrations usually prescribed for mEC-NB and TSB-CTV. These findings suggest that mEC-NB and TSB-CTV should be used at 36 degrees C for growth of VTEC O157 and that use of a nonselective pre-enrichment broth medium (i.e. TSB) together with a selective one (i.e. TSB-CTV or mEC-NB) is necessary for successful isolation of VTEC O157 from various specimens.     

Evaluation of universal preenrichment broth for the recovery of foodborne pathogens from milk and cheese

The use of universal preenrichment broth for the recovery of verotoxigenic Escherichia coli, Salmonella spp., and Listeria monocytogenes from milk and cheese was examined. Universal preenrichment broth supported the growth of low inoculum levels (10 cfu/ml) of these organisms in pure cultures and in mixed cultures containing higher levels of other pathogens or bacterial flora from raw milk. This medium also supported the recovery and growth of heat-injured Salmonella spp., L. monocytogenes, and verotoxigenic E. coli at inoculum levels of 10(2) cfu/ml to yield cell levels of 10(8) cfu/ml in pure cultures and at least 10(5) cfu/ml in the presence of high levels of known competitive pathogens or microflora of cheese samples after 24 h of incubation. Universal preenrichment broth performed better than Listeria enrichment broth in supporting the recovery and growth of heat-injured L. monocytogenes and equally as well as buffered peptone water or trypticase soy broth in supporting the growth of uninjured L. monocytogenes, Salmonella spp., and verotoxigenic E. coli. Coenrichment of these pathogens in universal preenrichment broth reduced the quantity of milk or cheese samples that were required for analysis and also reduced the cost and labor involved in preparing and processing separate preenrichment media.     

Membrane filter procedure for enumeration of Vibrio parahaemolyticus

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.     

Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk

Ovine milk that had been standardized to 6% fat was inoculated with Escherichia coli 405 CECT and Pseudomonas fluorescens 378 CECT at a rate of 10(6) and 10(7) cfu/ml, respectively, and treated with high hydrostatic pressure. Treatments consisted of combinations of pressure (300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). Inactivation (> 6 log cfu/ml) of both strains was observed at 50 degrees C for all pressures and treatment times. A similar level of inactivation occurred at > or = 450 MPa and 25 degrees C for E. coli and at > or = 400 MPa and 10 degrees C for P. fluorescens. Destruction was lowest at 10 degrees C for E. coli and at 25 degrees C for P. fluorescens. The test strain of E. coli was more baroresistance than was the P. fluorescens strain.     

A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Isoforms of myosin in growing muscles of ky (kyphoscoliotic) mice

The permeability of rat liver microsomes to glucose has been studied by using (14)C-labelled D-glucose and a light-scattering technique. 1) The microsomal intravesicular apparent isotope space for D-glucose (1mM; after 5 min incubation at 22 degrees C) was 2.34 microl/mg protein, i.e., approximately 72% of the apparent water space. 2) Efflux of [(14)C]D-glucose from microsomal vesicles pre-loaded as in 1) and measured by rapid Millipore filtration after dilution (100 fold) in a glucose-free medium revealed that 15 sec after dilution only 15% of intravesicular glucose was still retained by microsomes. 3) Osmotic behaviour of microsomes upon addition of D-glucose measured by a light-scattering technique revealed a glucose influx, saturable at [D-glucose] > 100 mM, and (partially) inhibited by pentamidine and cytochalasin B. Ascorbic acid, L-glucose and other monosaccharides and related compounds also permeated liver microsomes in a fashion similar to D-glucose. These data indicate the existence of a facilitative transport system(s) for glucose in the membrane of liver endoplasmic reticulum vesicles.