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PubMed2002

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system.

BACKGROUND: A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. METHODOLOGY: Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. RESULTS: The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. CONCLUSIONS: We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Acinetobacter↗
PubMed2005

Explaining the poor bacteriologic eradication rate of single-dose ceftriaxone in group a streptococcal tonsillopharyngitis: a reverse engineering solution using pharmacodynamic modeling.

OBJECTIVE: To explore pharmacokinetic factors underlying the poor bacteriologic eradication rate with a single 500-mg dose of ceftriaxone for streptococcal tonsillopharyngitis and to identify the minimum ceftriaxone dose required for effective treatment. METHODS: Population modeling techniques were applied to pharmacokinetic data derived from paired plasma and tonsil samples from 153 children to assess the contribution of pharmacokinetic variability to patients' responses to ceftriaxone. In addition, a Monte Carlo simulation was performed to determine (1) the amount of time that free ceftriaxone concentrations must exceed the minimum inhibitory concentration (MIC) of group A Streptococcus to achieve bacteriologic eradication and (2) the ceftriaxone dose required to maintain free drug concentrations above the target MIC for the requisite amount of time. Ceftriaxone MICs for group A Streptococcus were obtained from a previous trial, in which all MICs (n = 115) were < or = 0.064 mg/L; 33.9% were susceptible at < or = 0.016 mg/L, 66.4% were susceptible at 0.032 mg/L, and 1.7% were susceptible at 0.064 mg/L. RESULTS: Mean population pharmacokinetic parameters and their variances reflected substantial variability of clearance and half-life in the target population. Tonsillar ceftriaxone protein binding was 89.1%. The proportions of 1000 simulated patients with free ceftriaxone concentrations that exceeded MICs of 0.016 mg/L, 0.032 mg/L, and 0.064 mg/L at 24 hours were 71.7%, 65.4%, and 57.2%, respectively, and at 48 hours were 41.8%, 35.8%, and 28.6%, respectively. The amount of time that free ceftriaxone concentrations need to exceed MIC to achieve bacteriologic success was estimated to be 36 hours. Using this time criterion, two 500-mg doses of ceftriaxone separated by 18 hours should achieve a bacteriologic cure rate of approximately 95%. CONCLUSIONS: Pharmacokinetic variability and high ceftriaxone tonsillar protein binding explain the high microbiologic failure rate for a single 500-mg dose of ceftriaxone in group A streptococcal tonsillopharyngitis. Monte Carlo simulation suggests that a second dose administered 18 hours after the first will be required to achieve an acceptable bacteriologic cure rate.

Anti-Bacterial Agents↗
PubMed2006

Excisable cassettes: new tools for functional analysis of Streptomyces genomes.

The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli, the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces, allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int. The resulting strains are marker-free and contain an "attB-like" sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding polar effects if downstream genes are cotranscribed with the target gene. A set of cassettes was constructed to contain a hygromycin or gentamicin resistance gene flanked by the attL and attR sites. The initial constructions carrying convenient cloning sites allow the insertion of any other marker gene. We tested insertion and excision by inserting a cassette into orf3, the third gene of an operon involved in spiramycin biosynthesis. We verified that the cassette exerted a polar effect on the transcription of downstream genes but that, after excision, complementation with orf3 alone restored spiramycin production.

Attachment Sites, Microbiological↗
PubMedDate not supplied

[Antiviral activity of human recombinant gamma-interferon and recombinant hybrid protein of tumor necrosis factor-alpha-thymosin-alpha1 on models of herpes-virus and cytomegalovirus infection in vitro].

The conducted studies showed a certain efficiency of gene-engineering preparation of IFN-gamma and TNF-T in virus infections caused by herpes simplex virus 2 (HSV-2) and cytomegalovirus (CMV) in cell cultures of human embryo fibroblast (HEF). The drugs have no viricidal action. IFN-gamma, when used according to the treatment scheme in vitro, proved to be more effective versus TMF-T both in HSV-2 and in CMV. It inhibited significantly the HSV-2 reproduction within the dilution range of 1:50 to 1:500. It was also effective, when used in CMV, within the dilution range of 1:5000 and lower, whereas TNF-T was effective within the range of 1:500 and lower as well as in 0.1 multiple infection. A significantly higher effect was ensured when the drugs were used for prevention. In HSV-2, IFN-gamma inhibited the virus reproduction, like in the treatment scheme, within the dilution range of 1:50 to 1:500, whereas TNF-T was effective in the range of 1:50. In CMV, the drugs' effect, when used for prevention, was similar to that observed in the treatment scheme. The highest inhibition values were registered for HSV-2, when it was used 24 hours before infection (IFN-gamma--2.25 Ig, dilution range of 1:50; TNF-2--1.0 Ig, dilution range of 1:50). IFN-gamma and TNF-2 exert a synergic action on different stages of virus reproduction. A reliable additive effect was ensured in prevention made 4 hour before infection by IFN-gamma and TNF-T only in experimental CMV infection.

Antiviral Agents↗
PubMed1990

Microbial aggregates in anaerobic wastewater treatment.

The phenomenon aggregation of anaerobic bacteria gives an opportunity to speed up the digestion rate during methanogenesis. The aggregates are mainly composed of methanogenic bacteria which convert acetate and H2/CO2 into methane. Other bacteria are also included in the aggregates but their concentration is rather small. The aggregates may also be formed during acetogenesis or even hydrolysis but such aggregates are not stable and disrupt quickly when not fed. A two stage process seems to be suitable when high concentrated solid waste must be treated. Special conditions are necessary to promote aggregate formation from methanogenic bacteria but aggregates once formed are stable without feeding even for a few years. The structure, texture and activity of bacterial aggregates depend on several parameters: (1)--temperature and pH, (2)--wastewater composition and (3)--hydrodynamic conditions within the reactor. The common influence of all these parameters is still rather unknown but some recommendations may be given. Temperature and pH should be maintained in the range which is optimal for methanogenic bacteria e.g. a temperature between 32 and 50 degrees C and a value pH between 6.5 and 7.5. Wastewaters should contain soluble wastes and the specific loading rate should be around one kgCOD(kgVSS)-1 d-1. The concentration of the elements influences aggregate composition and probably structure and texture. At high calcium concentration a change in the colour of the granules has been observed. Research is necessary to investigate the influence of other elements and organic toxicants on maintenance of the aggregates. Hydrodynamic conditions seem to influence the stability of the granules over long time periods. At low liquid stream rates, aggregates may starve and lysis within the aggregates is possible which results in hollowing of aggregates and their floating. At high liquid stream rates the aggregates may be disrupted and washed out of the reactor as a flocculent sludge. Methanogenic bacterial aggregates have been successfully applied in many full scale installations, especially for sugar beet, potato, pulp and paper mill, and other soluble wastes. The UASB reactors used for these treatments are simple in construction and handling which result in rather low total costs. A further and wider application of UASB reactors and methanogenic aggregates for various industrial wastewaters is expected.

Bacteria, Anaerobic↗
PubMed2005

Neopeptide antibiotics that function as opsonins and membrane-permeabilizing agents for gram-negative bacteria.

We suggest a novel approach to enhancing antimicrobial drug action by utilizing engineered peptide conjugates. Our most potent conjugates, [fMLF]PMBN and [fMLF]PMEN, are nonapeptides derived from polymyxin B's (PMB's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Phe-Leu-Dab-Dab-Thr], where Dab is 2,4-diaminobutyric acid) and polymyxin E's (PME's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Leu-Leu-Dab-Dab-Thr]), respectively, attached to a linear tail comprised of formyl-Met-Leu-Phe (fMLF). The cyclic part binds to gram-negative lipopolysaccharides, rendering the bacterial outer membrane permeable to hydrophobic antibiotics. The tail confers chemotactic and opsonic activities upon the conjugates. These two activities appear to be the basis for the conjugates' antibacterial activities. The conjugates are 8 to 10 times less toxic than the parent PMB or PME antibiotics. Fourteen of 18 mice lethally challenged with erythromycin-resistant Klebsiella pneumoniae survived following intraperitoneal administration of erythromycin and [fMLF]PMBN, whereas erythromycin or the peptide conjugate alone had no effect. Moreover, the clearance of Klebsiella from blood was markedly enhanced by intravenous injection of the [fMLF]PMEN peptide conjugate compared to the clearance of the organism from the mice treated with buffer alone as a control and was similar to that achieved by the PME antibiotic. Blood clearance was also significantly enhanced by administration of PMEN either alone or in a mixture with fMLF, although the effect was less than that produced by the peptide conjugate. Since resistance to polymyxins, the parent molecules of the synthetic cyclic peptides, is rare, the emergence of bacteria resistant to the antimicrobial properties of the peptide conjugates may be precluded as well.

Animals↗
PubMed2026

Characterisation of metabolic burden in Pseudomonas putida reveals precursor limitation in heterologous lycopene production.

BACKGROUND: The introduction of heterologous pathways into microbial hosts often imposes a metabolic burden on the cell, arising from three major physiological constraint layers: competition for gene expression resources, limited precursor availability and flux distribution, and insufficient energy and redox supply. Although Pseudomonas putida KT2440 is considered a robust and metabolically versatile production host, it remains unclear which of these constraint layers primarily limits heterologous terpenoid production in this organism. Here, lycopene biosynthesis was used as a model system to systematically dissect these three potential sources of metabolic burden. RESULTS: A capacity-monitoring system revealed no clear reduction in transcriptional or translational capacity across the tested strains and cultivation conditions, indicating that general gene expression capacity was not the primary limiting factor. Instead, lycopene production depended strongly on promoter architecture and plasmid backbone, showing that regulatory design shaped pathway performance. Enhancing precursor supply by introducing a heterologous mevalonate (MVA) pathway substantially increased product titres, identifying precursor availability from the native MEP pathway as the dominant bottleneck. This conclusion was independently supported by exogenous mevalonate supplementation, which further increased lycopene accumulation but also revealed saturation at higher concentrations, suggesting that downstream pathway balance or enzyme capacity became limiting once precursor supply was relieved. Under controlled bioreactor conditions, lycopene titres increased from approximately 1&#xa0;mg/L to nearly 25&#xa0;mg/L, indicating that process conditions further modulate production performance, suggesting an additional contribution of process-dependent energy and redox constraints. CONCLUSION: Metabolic burden during heterologous lycopene production in P. putida is governed primarily by precursor availability rather than by limitations in general gene expression capacity. Regulatory properties of the vector system strongly influence pathway performance, while controlled cultivation conditions can further improve production by alleviating additional process-dependent constraints. Together, these findings provide a systematic framework for distinguishing constraint layers and guiding the optimisation of heterologous terpenoid production systems.

Lycopene↗
PubMed1986

Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor.

Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein. Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein. To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270). The analogs were produced in E. coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells. The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. The more extensive amino-terminal deletion analog rIL-1 (144-270) was inactive in all biological assays and failed to compete in the receptor binding assay. The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed. These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor. Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities. This amino-terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity.

Amino Acids↗
PubMed2002

Efficacy of bacterial removal from instrumented root canals in vitro related to instrumentation technique and size.

OBJECTIVE: This in vitro investigation assessed the efficacy of removing radioactively labeled bacteria from infected canals with 2 engine-driven rotary nickel titanium instrumentation techniques differing in sequence and apical enlargement size. STUDY DESIGN: A standard quantity of (3)H-thymidine-labeled Enterococcus faecalis (3.70 x 10(4) cpm, 2.0 x 10(7) colony-forming units) was used to inoculate the mesiobuccal canals of 50 extracted mandibular molars. The teeth were incubated for 5 days to allow infection of the surrounding dentin from the canals. Five of the teeth were used as controls to determine the number of cycles of irrigation and drying necessary to reduce the (3)H counts recovered from the canals to baseline levels. After this process, the unbound bacteria in the root canals of the remaining 45 teeth then were washed out with buffer until baseline levels of radioactivity were obtained. The mesiobuccal root of 1 of these 45 teeth was removed, decalcified, and digested, and the total radioactivity released from the root dentin was measured. Of the remaining 44 teeth, 22 then were instrumented with GT and Profile (Dentsply/Tulsa Dental Co, Tulsa, Okla) instruments to apical size #35 (group 1) and 22 teeth with Pow-R instruments (Moyco/Union Broach, York, Pa) to apical size #50 (group 2), in the presence of a standard quantity of phosphate-buffered saline solution placed in the canal. After instrumentation, the medium from each canal was collected with paper points and its radioactivity was counted with liquid scintillation spectrometry. RESULTS: The mean (3)H level recovered with instrumentation of canals in group 1 was 75 cpm (+/- 29, standard deviation) and in group 2 was 123 cpm (+/- 50, standard deviation). A 2-tailed Mann-Whitney test indicated that the radioactivity of samples from group 2 was significantly higher than that of samples from group 1. CONCLUSION: The results suggested that instrumentation to an apical size of #50, as performed with the Pow-R instruments, was more effective in debriding infected root canals than instrumentation to an apical size of #35, as performed with the GT and Profile instruments.

Colony Count, Microbial↗
PubMed1984

The impact of new technologies on vaccine development.

The ability to move genetic determinants between species using in vitro gene-manipulation techniques has opened up new approaches to vaccine development. This has rapidly grown into an exciting area of research in both academic and industrial laboratories. There are numerous scientific challenges which require multidisciplinary teams to solve problems in creating new immunogens. This has challenged our existing knowledge about protein structure and conformation, microbial pathogenicity and the immune system. Recombinant-DNA techniques are invaluable as tools of analysis and antigen production. The surface of micro-organisms can also be minutely explored with the use of synthetic peptides and monoclonal antibodies. Nevertheless, these new technologies do not allow us to circumvent the need for detailed understanding of pathogens and the disease process. What is apparent from the work carried out so far is that there are few easy answers to vaccine development and it is not realistic to expect rapid solutions to these problems. As there are many potential targets for constructing novel vaccines for both human and animal diseases, it is helpful to establish some priorities. There is a tendency to look at the existing effective vaccines and simply direct research at producing them more economically or with enhanced safety and stability. The advantage of this approach is that considerable background work will have already been carried out establishing the basis for the application of recombinant DNA techniques. However, this can also lead to conflicts (often within the same institute or company) between the new and old technologies. This could be to the detriment of the new technologies which are still only partly developed and may not be good enough yet to compete with existing vaccines in cost or efficacy. The more ambitious, and eventually more rewarding, approach is to attempt to develop new vaccines where none had existed before. There is a vast untapped market, especially in the parasitic diseases, but the scientific problems may be considerable and much more background work is likely to be necessary. Indeed, most of the work in this area is more accurately referred to as basic research rather than vaccine development as totally new, effective vaccines are still some way off. Having directed research towards a specific organism or disease there are still many options available as to the scientific strategy to adopt. As discussed in this review it may be possible to consider subunits, synthetic antigens and live (attenuated or heterologous) organisms as possible vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvants, Immunologic↗
PubMedDate not supplied

Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant).

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.

Directed Molecular Evolution↗
PubMed2005

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria.

BACKGROUND: In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. RESULTS: The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. CONCLUSION: We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.

Anti-Bacterial Agents↗