Survival of selected bacterial species in sterilized activated carbon filters and biological activated carbon filters.

The survival of selected hygienically relevant bacterial species in activated carbon (AC) filters on a bench scale was investigated. The results revealed that after inoculation of the test strains the previously sterilized AC absorbed all bacteria (10(6) to 10(7)). After a period of 6 to 13 days without countable bacteria in the effluent, the numbers of Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida increased up to 10(4) to 10(5) CFU/ml of effluent and 10(6) to 10(7) CFU/g of AC. When Klebsiella pneumoniae and Streptococcus faecalis were used, no growth in filters could be observed. The numbers of E. coli, P. aeruginosa, and P. putida, however, decreased immediately and showed no regrowth in nonsterile AC from a filter which had been continuously connected to running tap water for 2 months. Under these conditions an autochthonous microflora developed on the carbon surface which could be demonstrated by scanning electron microscopy and culturing methods (heterotrophic plate count). These bacteria reduced E. coli, P. aeruginosa, and P. putida densities in the effluent by a factor of more than 10(5) within 1 to 5 days. The hypothesis that antagonistic substances of the autochthonous microflora were responsible for the elimination of the artificial contamination could not be confirmed because less than 1% of the isolates of the autochthonous microflora were able to produce such substances as indicated by in vitro tests. Competition for limiting nutrients was thought to be the reason for the observed effects.     

Effects of activated carbon and bacteriostatic filters on microbiological quality of drinking water

Three activated carbon filters for point-of-use water treatment were tested in laboratory and field studies for chemical removal and microbiological effects on water. All removed free available chlorine in municipally treated water to below the limit of detection, but removed only about 50 to 70% of the total available chlorine and 4 to 33% of the total organic carbon. Standard plate count bacteria in the effluent increased steadily with time for 3 weeks and remained elevated over the 8-week period of the study. Total coliform bacteria were found to persist and proliferate on the filters for several days after transient contamination of the influent water. Silver-containing activated carbon filters suppressed total coliform but not total bacterial growth. Pseudomonas aeruginosa was recovered from the effluents of all filters at some time during the tests.     

Effects of activated carbon and bacteriostatic filters on microbiological quality of drinking water.

Three activated carbon filters for point-of-use water treatment were tested in laboratory and field studies for chemical removal and microbiological effects on water. All removed free available chlorine in municipally treated water to below the limit of detection, but removed only about 50 to 70% of the total available chlorine and 4 to 33% of the total organic carbon. Standard plate count bacteria in the effluent increased steadily with time for 3 weeks and remained elevated over the 8-week period of the study. Total coliform bacteria were found to persist and proliferate on the filters for several days after transient contamination of the influent water. Silver-containing activated carbon filters suppressed total coliform but not total bacterial growth. Pseudomonas aeruginosa was recovered from the effluents of all filters at some time during the tests.     

Growth and persistence of pathogens on granular activated carbon filters.

Three enteric pathogens Yersinia enterocolitica O:8, Salmonella typhimurium, and enterotoxigenic Escherichia coli, were examined for their ability to colonize granular activated carbon (GAC) in pure cultures and in the presence of autochthonous river water organisms. All three organisms readily colonized sterile GAC and maintained populations of ca. 10(5) to 10(7) CFU g-1 for 14 days when suspended in sterile river water. Exposure of pathogen biofilms on GAC to unsterile river water resulted in a gradual decline in pathogens on the carbon (0.08 to 0.14 log day-1). When pathogens were introduced to sterile GAC in the presence of heterotrophic plate count organisms, they attached at levels similar to those in the pure cultures and then decreased (0.10 to 0.22 log day-1). When added with heterotrophic plate count bacteria to GAC supporting a mature biofilm of native river water bacteria, they attached at a lower level (1.0 X 10(4) to 4.6 X 10(4) CFU g-1) and decreased at a more rapid rate (0.11 to 0.70 log day-1).     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2.

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.     

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.     

Autoradiographic study of the localization and evolution of growth zones in bacterial colonies.

Incorporation of [3H]leucine in the bacteria of 18 to 48 h-old colonies of Pseudomonas aeruginosa, Pseudomonas putida, Bacillus thuringiensis, Staphylococcus aureus and Escherichia coli enabled the localization of bacterial multiplication sites by means of autoradiography of sagittal sections. In colonies where fast diameter expansion occurred, all the bacteria from the peripheral corona contributed to peripheral growth; in colonies where the expansion was slower, the growth rate of the bacteria in this region was heterogeneous. Besides this peripheral growth, a central region of bacterial multiplication was always found, but with variable localization and extension. In aerobic species, such as P. aeruginosa and P. putida, the central growth site was limited to the zone of oxygen penetration into the bacterial mass. However, in facultatively anaerobic species, bacterial multiplication dependend on nutrient supply. For 48 h-old colonies of S. aureus, a more complex localization of growth seemed to be affected simultaneously by nutrient penetration and accumulation of toxic substances.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.     

The use of copper and silver in carbon point-of-use filters for the suppression of Legionella throughput in domestic water systems

Aims: To evaluate throughput of seeded Legionella pneumophila bacteria in domestic point‐of‐use filters. Methods and Results: The filters were challenged with tap water seeded with Leg. pneumophila. After multiple challenge events (4·25 × 10¹¹ CFU per filter), the levels of Legionella were lower in the effluent from the filter containing both copper and silver (mean 4·48 × 10³ CFU ml^?1) than in the effluent from the filter containing copper only (1·26 × 10⁴ CFU ml^?1; P < 0·001). After a single challenge event of approx. 5 × 10⁹ CFU L. pneumophila per filter, there was no significant difference between the levels of Legionella in the effluents from a carbon filter containing copper and a carbon filter with no metals (mean 6·87 × 10² and 6·89 × 10² CFU ml^?1, respectively; P = 0·985). Conclusions: Legionella was detected in filter effluent up to 6 weeks after being challenged, indicating that while filters may reduce the levels during an initial contamination event, the exposure is extended as the accumulated bacteria slough off over time. Significance and Impact of the Study: This study has provided an understanding of the response of Legionella to the use of silver and copper in domestic point‐of‐use carbon filters.     

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Molecular analysis of the Pseudomonas aeruginosa regulatory genes ptxR and ptxS.

We have previously described two Pseudomonas aeruginosa genes, ptxR, which enhances toxA and pvc (the pyoverdine chromophore operon) expression, and ptxS, the first gene of the kgu operon for the utilization of 2-ketogluconate by P. aeruginosa. ptxS interferes with the effect of ptxR on toxA expression. In this study, we have utilized DNA hybridization experiments to determine the presence of ptxR and ptxS homologous sequences in several gram-negative bacteria. ptxR homologous sequences were detected in P. aeruginosa strains only, while ptxS homologous sequences were detected in P. aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens. Using Northern blot hybridization experiments and a ptxS-lacZ fusion plasmid, we have shown that P. aeruginosa ptxR and ptxS are expressed in P. putida and P. fluorescens. Additional Northern blot hybridization experiments confirmed that ptxS is transcribed in P. putida and P. fluorescens strains that carried no plasmid. The presence of a PtxS homologue in these strains was examined by DNA-gel shift experiments. Specific gel shift bands were detected when the lysates of P. aeruginosa, P. putida, and P. fluorescens were incubated with the ptxS operator site as probe. kgu-hybridizing sequences were detected in P. putida and P. fluorescens. These results suggest that (i) ptxR is present in P. aeruginosa, while ptxS is present in P. aeruginosa, P. putida, and P. fluorescens; (ii) both ptxR and ptxS are expressed in P. putida and P fluorescens; and (iii) a PtxS homologue may exist in P. putida and P. fluorescens.     

One-step cloning system for isolation of bacterial lexA-like genes.

A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.     

Occurrence, transfer and mobilization in epilithic strains of Acinetobacter of mercury-resistance plasmids capable of transformation

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.     

Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.