Effect of introduced phthalate-degrading bacteria on the diversity of indigenous bacterial communities during di-(2-ethylhexyl) phthalate (DEHP) degradation in a soil microcosm.

Four previously isolated di-butyl-phthalate (DBP) degraders were tested for their abilities to degrade di-(2-ethylhexyl) phthalate (DEHP). In aqueous medium supplemented with 100mg/l of DEHP, both isolate G1 and Rhodococcus rhodochrous G2 showed excellent degradative activity; in three days they were able to degrade more than 97% of the added DEHP. Rhodococcus rhodochrous G7 degraded 32.5% of the added DEHP and Corynebacterium nitrilophilus G11 showed the least amount of DEHP degradation. The addition of surfactant Brij 30 at 0.1x critical micelle concentration (2mg/l) significantly improved DEHP degradation by Rhodococcus rhodochrous G2 (more than 90% of the added DEHP was degraded within 24 hours), but slightly inhibited the degradation of DEHP by the isolate G1 and Rhodococcus rhodochrous G7. Based on the 16S rDNA sequence data, isolate G1 was identified as Gordonia polyisoprenivorans. Soil inhibited DEHP degradation by G. polyisoprenivorans G1; fourteen days after a second addition of DEHP, 11.5% of the total added DEHP (i.e., 243.4 microg/g soil) remained detectable. Changes in the bacterial community were monitored using denaturing gradient gel electrophoresis (DGGE) and respective dendrogram analysis. It is clear that DEHP and DEHP plus G. polyisoprenivorans G1 substantially affected the bacterial community structure in the soils. However, as the population of indigenous DEHP degraders increased in the DEPH-treated soil, its bacterial communities resembled those in the DEHP plus G. polyisoprenivorans G1-inoculated soil by Day 17.

Evaluation of Colilert-18 for the detection of coliforms and Escherichia coli in tropical fresh water.

AIMS: To evaluate the suitability of Colilert-18 in detecting Escherichia coli and total coliforms in tropical freshwater samples. METHODS AND RESULTS: Target organisms were isolated from yellow-fluorescent and yellow wells of Colilert-18/Quanti-Tray using m-TEC agar and m-ENDO LES agar respectively. All the selected isolates were first identified based on their fatty acid methyl ester profile. Isolates showing contradictory results to that of the Colilert-18 procedure were re-identified using API 20E strips. A total of 357 isolates, 177 from yellow-fluorescent wells and 180 from yellow wells, were identified. CONCLUSIONS: The false-positive and -negative rates for E. coli detection using Colilert-18 were 36.4% and 11%, respectively, while for coliform detection the false-positive rate was 10.3%. SIGNIFICANCE AND IMPACT OF THE STUDY: The high false-positive rate of Colilert-18, tempers its value for E. coli detection when used for tropical freshwater samples.

Degradation of di-butyl-phthalate by soil bacteria.

Twelve Gram-positive phthalate ester degraders were isolated from soil. Using Biolog GP2 plates, eight of them were identified as belonging to the Corynebacterium-Mycobacterium-Nocardia group, while the remaining four were unidentifiable. When cultured in the presence of di-butyl-phthalate (DBP) in basal salts solution, five of these isolates accomplished more than 90% of DBP degradation within 48 h (fast group), three were placed in the medium group, and the remaining four were placed in the slow group which caused less than 30% of DBP degradation within the same period of time. A 420 bp DNA fragment was amplified from six isolates and none of them fell within the slow group. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, 83% and 91% similarities were evident in the nucleotide and amino acid sequences, respectively. However, no correlation between cell surface hydrophobicity and phthalate degradation ability was evident. Six surfactants (Brij 30, Brij 35, Tergitoltype NP-10, Triton N-101, Triton X-100 and SDS) were tested for their abilities to increase degradation rate. When added at the critical micellar concentration (CMC), they all displayed strong growth inhibition against the three bacteria tested, with Brij 30 been the least toxic to isolates G2 and G11, and Brij 35 had the least inhibitory effect for G1. When half the CMC of Brij 30 was incorporated into the basal salts, the inhibitory effect on DBP degradation remained. Soil helped to minimize surfactant toxicity of surfactant and increase the degradation potential of some of the test bacteria. When DBP-amended soil had been aged for three months, decreases in bioavailability were observed but the effect varied tremendously between different organisms. For isolates G1, G2, G5, G7 and G17 the aging effects were almost non-exist. The present study indicates that selection of a suitable degrader may minimize the undesired effect of aging on bioremediation process.

Simultaneous enumeration of different bacteria using reverse sample genome probing technique.

Quantitative reverse sample genome probing (RSGP) with lambdaDNA as an internal standard was used to enumerate the total numbers of Rhizobium sp. CCRC 13560, Rhizobium meliloti CCRC 13516 and Bradyrhizobium sp. CCRC 13585. K(lambda)/Kx ratios varied between the three species but also in response to the amounts of lambdaDNA or genomic DNA used in the labeling mixture or fixed upon the membrane. Comparative enumerations of pure cultures revealed higher counts using genomic probing as compared with growth-based colony forming units (CFU; 3.4+/-1.7-fold higher for R. meliloti, 6.4+/-7.8-fold higher for Rhizobium sp. and 0.34+/-0.17-fold higher for Bradyrhizobium sp.). In mixed cultures, the estimated cell numbers using genomic probing were 126+/-172-, 85+/-83- and 4.0+/-3.4-fold higher (same respective order) than the growth-based assay. By replacing the klambda/kx ratio with k'lambda/k'x (slope from signal intensity of differently diluted lambdaDNA/slope from signal intensity of differently diluted target DNAxf(x)/flambda), significant improvement in the accuracy of the estimation was achieved. The calculated cell numbers via the genomic probe technique were 0.99+/-0.13-, 1.25+/-0.23- and 0.18+/-0.11-fold higher than the respective CFUs in pure cultures of R. meliloti, Rhizobium sp. and Bradyrhizobium sp. In samples containing mixed populations, the estimated numbers from genomic probing were 1.25+/-0.51-, 45.9+/-14.8- and 0.27+/-0.07-fold higher than the CFU-derived cell count (same respective order).

Decoloration of azo dyes by three whiterot fungi: influence of carbon source.

Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high nitrogen medium with glucose, could decolorize two azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). When starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12MM NH4Cl, whether or not veratric acid was present. In medium containing 1.2MM NH4Cl and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2MM NH4Cl, decoloration by the local isolate was enhanced but not that by the other strains.

In vitro and in situ survivals of bacterial populations added to fresh water environments.

The fate of Aeromonas hydrophila, Alcaligenes denitrificans, Vibrio cholerae non-01, Pseudomonas putida and four different isolates of Escherichia coli in fresh river water were assessed by using different microcosms (i.e., membrane diffusion chamber and Erlenmeyer flask). When water samples were incubated at 16 +/- 1 degrees C, the differences in extent of survival among test bacteria were in general not significant. If the incubation temperature was raised to 29 +/- 1 degrees C, in the in situ studies, none of the added bacterial population could be detected by Day 3. In the in vitro studies, two of the four E. coli tested remained detectable by Day 3. Similarly, populations of the introduced A. hydrophila, P. putida and A. denitrificans were still detectable by Day 5. In general, all test bacteria survived better under low incubation temperature, regardless of whether the experiments were carried out under in vitro or in situ conditions. The results clearly indicated that when studying the fate of the introduced bacteria in the aquatic environment, in situ study was definitely required, especially in the summer time.

Detection of Listeria monocytogenes by non-radioactive RNA probe and polymerase chain reaction.

A HindIII fragment, 651-base-pair, from the listeriolysin 0 gene of Listeria monocytogenes was cloned into pGEM4Z. Radioactive 32P labeled and non-radioactive digoxigenin labeled RNA probes were made by in vitro transcriptions. These probes hybridized only to L. monocytogenes strains. Polymerase chain reaction was performed by using a 22-mer and a 23-mer oligonucleotide primer, and segment of about 700-base-pair was amplified only in L. monocytogenes strains and this band was visualized by hybridization with the non-radioactive probe.

Removal of copper ion by Pseudomonas spp.

Copper-resistant Pseudomonas sp. 41Y, Pseudomonas pseudomallei 13-1 and Pseudomonas aeruginosa 7 were used in the present study. When the latter two organisms were added to copper-containing 1/3 strength Tryptic Soy Broth, more than 99.5% of the copper ion was removed from the medium within 24 h. If copper solution was added to hog waste slurry, a reduction in the copper ion concentration could be detected only when the added bacteria started to grow in it, whereas in a mineral medium supplemented with glycerol-2-phosphate, both bacteria could remove about 50% of the copper ion from the medium within 24 h. When cell suspension of Pseudomonas sp. 41Y was autoclaved, no copper ion removal was observed. Different incubation temperatures, including 30 degrees C, 37 degrees C and 45 degrees C, had no effect on the percent of copper ion removed by both Pseudomonas sp. 41Y and P. pseudomallei 13-1. On the other hand, if the pH value of the solution was lowered from 8.2 to 6.0, there was a drastic decrease in copper removal. A similar reduction of copper ion removal ability was also observed with the addition of lead ion. When cells of Pseudomonas sp. 41Y were embedded in sodium alginate, there was a decrease in its ability to remove copper ion as compared to the free-living cells.

Incidence and characterization of Listeria monocytogenes in foods available in Taiwan.

A variety of foods were examined for the incidence of Listeria monocytogenes, and the bacterial isolates were further characterized. L. monocytogenes was selected on LiCl-phenylethanol-moxalactam agar after enrichments and identified by several biochemical, mobility, and CAMP tests. L. monocytogenes was isolated from 58.8% of pork samples, 50% of chicken carcasses, 38% of turkey parts, 34% of frozen semiready foods, 24% of beef steaks, 12.2% of vegetables, 10.5% of seafoods, and 4.4% of frozen dim sum but was not found in the Chinese pickles and fermented milks. Isolates from seafoods, turkey parts, and beef samples had higher hemolytic activity than those from other samples. The isolates were highly susceptible to ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, kanamycin, neomycin, novobiocin, penicillin, and streptomycin. About 14.5% of the isolates were resistant to methicillin, and 14.5% were resistant to tetracycline. The majority of the isolates from turkey parts and beef steaks were serotype 1, and those from chicken and pork samples were serotype 4 and others. Hemolytic activity, methicillin susceptibility, and serotype distribution of the isolates from domestic and imported food samples were significantly different. The results suggest the presence of food- or geography-specific L. monocytogenes strains.

Survival of genetically engineered Escherichia coli in natural soil and river water.

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased.

Effect of root agglutinin on microbial activities in the rhizosphere.

A total of 220 bacterial isolates were obtained from pea rhizosphere and nonrhizosphere samples. Of these samples, 100 isolates were chosen randomly to test for their agglutinative reaction against pea root exudate. The percentage of positive agglutination of bacteria isolated from the nonrhizosphere sample was significantly lower than that of bacteria isolated from the rhizosphere sample. Moreover, this agglutinative reaction could not be blocked either by treating the bacterial cells or root exudate with different carbohydrates before they were mixed or by boiling the root exudate first. Bacteria that could be agglutinated by pea root exudate followed the downward growth of the pea root through the soil profile. The greater abilities of such bacteria to colonize the pea rhizosphere were indicated by their higher rhizosphere-colonizing (rhizosphere/nonrhizosphere) ratios, whether the bacteria were added alone or together with nonagglutinating bacteria. However, bacteria did show different agglutinative reactions toward root exudates obtained from different plants.

Survival of Yersinia enterocolitica in the environment.

When Yersinia enterocolitica was introduced into soils (or physiological saline), very little decrease in the population was observed throughout the test period. If the soil was allowed to air dry slowly, only 0.1% (2.8 x 10(3) colony forming units/g of soil) of the original population added still remained viable by day 10. On the other hand, the introduced organisms disappeared rapidly in river water but their longevities could be extended significantly if a eucaryote inhibitor was added to the river water or the river water was passed through a 0.8-micron membrane filter to remove eucaryotic predators. Furthermore, the rapid decrease of the Yersinia population coincided with an increase in numbers of protozoans. However, when Yersinia was added to filter-sterilized river water or when small numbers of the organism, below the threshold level believed necessary for active predation to occur, were added to the river water, no response in predators was observed; nevertheless, the population of Yersinia still showed a continued decline. When the organism was introduced into sephadex-treated river water or groundwater, its survival improved significantly compared with its survival in nontreated water samples. Low ambient temperature dramatically increased its ability to survive in the aquatic environment. It is concluded that, in addition to the temperature factor, the longevity of Y. enterocolitica in river water is chiefly regulated by predators and toxin producers.

Factors affecting the survival of pathogenic bacteria in subtropical river water.

The present study was carried out to determine the factors causing the disappearance of introduced Salmonella enteritidis, Staphylococcus aureus, and Vibrio cholerae in natural river water. When large numbers of the above organisms (final level 10(7)-10(8) CFU per ml) were added to river water, the decline of their numbers coincided with increasing numbers of protozoa. Furthermore, their survival was improved dramatically if the river water was amended with a eucaryote inhibitor. After comparing the survivals of the test organisms in physiological saline, filtered river water, and in dialysis tubes immersed in river water, the results suggested that for Salmonella, predation was the most significant factor which caused the decline of its population in natural river water; starvation and toxic materials played lesser roles. Staphylococcus and Vibrio were also vulnerable to predation and toxic materials have molecular weights larger than 12,000. In the natural environment, these three factors together could cause rapid disappearance of the introduced microorganisms.

Survival of pathogenic bacteria in environmental microcosms.

The survival times of Salmonella enteritidis, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae in natural soil and river water samples were monitored by using various differential media. S. aureus and V. cholerae failed to survive in samples of soil, groundwater, and river water with various degrees of eutrophication. The population of the introduced S. enteritidis remained fairly constant in all three samples of soils tested. In water samples, numbers of S. enteritidis and P. aeruginosa showed an initial rapid decline followed by a much lower rate of decrease. The results indicated that some allochthonous microorganisms, because of their insensitivity to various biotic and abiotic stresses, might persist for a long time in the environment and become a serious threat to public health.

Mineral soils as carriers for Rhizobium inoculants.

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4 degrees C than at higher temperatures, but ca. 15% of the cells were viable at 37 degrees C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35 degrees C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25 degrees C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.