The effect of tetracycline on the structure of the bacterial community in a wastewater treatment system and its effects on nitrogen removal.

This study examined the impact of tetracycline at two environmentally relevant concentrations (1 µg/L and 10 µg/L) and one synthetically high concentration (500 µg/L) on the structure and function of the microbial community from the secondary treatment process of a municipal wastewater treatment plant (WWTP). Specifically, this study examined whether the introduction of tetracycline into bench scale reactors at two different replacement volume rates would cause a shift in the composition profile of the bacterial community. Furthermore concentrations of ammonia, nitrate/nitrite and total Kjeldahl nitrogen were monitored to examine the effect of the antibiotic on ammonia and nitrogen removal. At the low volume replacement rate, tetracycline was observed to have a positive impact on nitrogen removal. Total Kjeldahl nitrogen concentrations were also observed to decrease suggesting a role for tetracycline as a carbon source. However, at the higher volume replacement rate, the removal of ammonia and nitrogen were not significantly different from reactors that did not contain tetracycline. Over time, the bacterial composition profiles changed under all the conditions studied, however, the bacterial composition profiles appeared to be more influenced by the replacement volume rate than the presence of tetracycline even at concentrations many times higher than environmentally relevant amounts.

Effects of tetracycline and ibuprofen on the relative abundance of microbial eukaryotic and bacterial populations in wastewater treatment.

The activated sludge process in a wastewater treatment plant (WWTP) relies on the activity of microbes to reduce the organic and inorganic matter and produce effluent that is safe to discharge into receiving waters. This research examined the effects of the non-steroidal anti-inflammatory drug ibuprofen and the antibiotic tetracycline on the relative abundance and composition of eukaryotes and bacteria in the microbial population present in activated sludge from a WWTP. The current investigation was designed to observe the impact of these contaminants, at low (environmentally relevant concentrations) as well as high concentrations of the drugs. Using 16S and 18S rRNA gene primer sets and quantitative polymerase chain reaction, the abundance of each population was monitored as well as the relative ratio of the two populations under the various conditions. It was found that current environmentally relevant concentrations of ibuprofen (100 ng/mL) stimulated eukaryotic growth but higher concentrations (2,000 ng/mL, 100,000 ng/mL) reduced their numbers significantly especially in the presence of tetracycline. Finally using denaturing gradient gel electrophoresis, some of the more abundant eukaryotes were identified and it was noted that high ibuprofen and tetracycline concentrations favoured the abundance of some genera.

Automated image analysis of Euglena gracilis Klebs (Euglenophyta) for measuring sublethal effects of three model contaminants.

The short-term impacts of atrazine (herbicide), tributyltin (organometal) and copper on the behaviour of Euglena gracilis Klebs (Euglenophyta) were assessed. First, the ECOTOX automated image analysis system was used, which measured swimming velocity, cell shape, percentage of cells swimming upwards, and randomness of swimming. Next, visual observation by microscopy was used to measure percentage of cell motility and cell shape. Behavioural changes can be used as an indicator of stress in less than 24 h, potentially making them suitable for inclusion in early-warning systems for water quality. Findings indicate that E. gracilis is a very sensitive organism to copper, showing inhibition of motility with visual observation at 0.8 µmol/L within 1 h. The image analysis system was in general less sensitive than visual observation for detecting behavioural changes after incubation in copper. In contrast, after exposure to organic contaminants atrazine and tributyltin, the ECOTOX system detected small changes in the number of cells swimming upwards (antigravitactic behaviour) at higher concentrations.

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

Effect of chemical and physical parameters on a pulp mill biotreatment bacterial community.

The bacterial community composition in an activated sludge plant treatment from a bleached kraft pulp mill was monitored over a period of 209 days. Using DGGE and terminal-Restriction Fragment Length Polymorphism (t-RFLP) analysis we generated community DNA fingerprints over the time period. Both methods produce fingerprints that can be used to monitor stability in the system and generate fragments that can be associated with bacterial taxa. Chemical and physical parameters were also collected during that same time frame. We found a number of significant correlations with influent variables such as temperature, chemical oxygen demand (COD), Biochemical oxygen demand (BOD) and chloroform concentrations suggesting that these were the most likely parameters to influence the bacterial community structure. In addition several taxa correlated to important performance indicators such as COD/BOD removals and SVI. Multivariate analysis also confirmed the strong links between taxa variation and temperature, nutrient loads, chloroform and also one class of filaments. Establishing the identity of these taxa and their ecological preferences will greatly enhance our understanding and management of biological treatment systems.

Brain proton magnetic resonance spectroscopy and imaging in children exposed to cocaine in utero.

OBJECTIVE: The effects of prenatal cocaine exposure have been examined using neurobehavioral and brain structural evaluations; however, no study has examined the effects of prenatal cocaine on brain metabolism. Proton magnetic resonance spectroscopy ((1)H-MRS) is a noninvasive method to examine the biochemistry of various brain regions. The purpose of this study was to examine the possible neurotoxic effects of prenatal cocaine exposure on the developing brain using (1)H-MRS. METHODS: Cocaine-exposed children (n = 14) and age-matched unexposed control participants (n = 12) were evaluated with MRI and localized (1)H-MRS. Metabolite concentrations of N-acetyl-containing compounds (NA), total creatine (Cr), choline-containing compounds, myoinositol, and glutamate + glutamine were measured in the frontal white matter and striatum. RESULTS: Despite an absence of structural abnormalities in either group, children exposed to cocaine in utero had significantly higher Cr (+13%) in the frontal white matter. NA, primarily a measure of N-acetyl aspartate and neuronal content, was normal in both regions examined by (1)H-MRS. Normal NA suggests no significant neuronal loss or damage in the 2 brain regions examined in children exposed to cocaine prenatally. CONCLUSIONS: Consistent with findings in abstinent adult cocaine users, we found increased Cr in the frontal white matter, with normal NA in children exposed to cocaine. These findings suggest the need to investigate further possible abnormalities of energy metabolism in the brain of children exposed to cocaine in utero. In addition, this study demonstrates the feasibility of using (1)H-MRS to investigate the effects of prenatal drug exposure on the developing brain.

Differential modulation of sodium channel gating and persistent sodium currents by the beta1, beta2, and beta3 subunits.

Brain sodium channels are complexes of a pore-forming alpha subunit with auxiliary beta subunits, which are transmembrane proteins that modulate alpha subunit function. The newly cloned beta3 subunit is shown to be expressed broadly in neurons in the central and peripheral nervous systems, but not in glia and most nonneuronal cells. Beta1, beta2, and beta3 subunits are coexpressed in many neuronal cell types, but are differentially expressed in ventromedial nucleus of the thalamus, brain stem nuclei, cerebellar Purkinje cells, and dorsal root ganglion cells. Coexpression of beta1, beta2, and beta3 subunits with Na(v)1.2a alpha subunits in the tsA-201 subclone of HEK293 cells shifts sodium channel activation and inactivation to more positive membrane potentials. However, beta3 is unique in causing increased persistent sodium currents. Because persistent sodium currents are thought to amplify summation of synaptic inputs, expression of this subunit would increase the excitability of specific groups of neurons to all of their inputs.

Laminin and heparan sulfate proteoglycan mediate epithelial cell polarization in organotypic cultures of embryonic lung cells: evidence implicating involvement of the inner globular region of laminin beta 1 chain and the heparan sulfate groups of heparan sulfate proteoglycan.

The extracellular matrix and in particular the basement membrane (BM) play an important role in the induction of organotypic rearrangement of cells in culture. This process involves cell aggregation, sorting into epithelial and mesenchymal components, epithelial cell polarization, and lumen formation. In this study, a combination of laminin (LM) and heparan sulfate proteoglycan (HSPG), two major BM constituents, induced organotypic rearrangement of embryonic mouse lung cells. In the absence of LM/HSPG supplementation, the cells sorted into epithelial and mesenchymal compartments but epithelial cell polarization and lumen formation did not occur. Neither LM nor HSPG alone could trigger this process. Synthetic peptide F-9, representing an amino acid sequence from the inner globular region of the laminin beta1 chain (RYVVLPRPVCFEKGMNYTVR) induced organotypic cell rearrangement when substituted for LM. Exogenous LM as well as peptide F-9 were localized at the epithelial-mesenchymal interface of organotypic cultures, where a BM-like structure is formed de novo. Organotypic cell rearrangement was blocked by heparin, heparan sulfate, or antibodies against peptide F-9. Binding assays indicated that peptide F-9 interacts with HSPG but not with LM or type IV collagen. Preincubation of embryonic lung cells with peptide F-9 resulted in a significant increase in cell attachment to HSPG but not to other major BM constituents. These findings suggest that the interaction between LM and BM HSPG is critical for the development of epithelial cell polarization and lumen formation. This interaction occurs at the epithelial-mesenchymal interface and is mediated by a site in the LM molecule represented by peptide F-9 and the heparan sulfate groups of HSPG.

Amphiregulin in lung branching morphogenesis: interaction with heparan sulfate proteoglycan modulates cell proliferation.

Epithelial and mesenchymal cells isolated from mouse embryonic lungs synthesized and responded to amphiregulin (AR) in a different fashion. Mesenchymal cells produced and deposited 3- to 4-fold more AR than epithelial cells, proliferated in the presence of exogenous AR, and their spontaneous growth was blocked by up to 85% by anti-AR antibodies. In contrast, epithelial cells exhibited a broad response to this growth regulator factor depending on whether they were supplemented with extracellular matrix (ECM) and whether this ECM was of epithelial or mesenchymal origin. AR-treated epithelial cells proliferated by up to 3-fold in the presence of mesenchymal-deposited ECM, remained unchanged in the presence of epithelial-deposited ECM, and decreased in their proliferation rate below controls in the absence of ECM supplementation. This effect was abolished by treatment with the glycosaminoglycan-degrading enzymes heparinase and heparitinase suggesting the specific involvement of heparan sulfate proteoglycan (HSPG) in AR-mediated cell proliferation. In whole lung explants, branching morphogenesis was inhibited by antibodies against the AR heparan sulfate binding site and stimulated by exogenous AR. Since during development, epithelial cells are in contact with mesenchymal ECM at the tips of the growing buds and alongside the basement membrane, focal variations in the proportion of epithelial and mesenchymal HSPG will focally affect epithelial proliferation rates. Therefore, AR-HSPG interaction may underlie the process of branching morphogenesis by inducing differential cell proliferation.

Developmental testing.

Pediatricians play a central role in monitoring the development of infants and children during the course of providing well child care. Parents turn to pediatricians for help in determining whether their child has a temporary lag in development, a serious delay or disorder, or a significant behavior problem that should be addressed. With the passage of PL 99-457, pediatricians also play a key role in referring children at risk to early intervention services. By employing a strategy of developmental surveillance, with periodic developmental screening, the pediatrician can determine when a child should be referred for more extensive developmental or psychological testing, which will aid in the process of diagnosis and treatment of developmental disabilities and behavioral disturbances. Knowledge of the screening and testing measures used commonly, as well as their limitations, will result in more accurate interpretation of the data derived from such measures. Once delays are diagnosed and treatment is initiated, repeated assessments over time will serve to identify areas in need of continuing intervention while indicating gains made in specific areas of developmental functioning. Throughout this process, the pediatrician's role as advocate for the child and family serves as a bridge to other professionals and services, with the ultimate goal of facilitating the optimal development of the child.

Two separate domains of laminin promote lung organogenesis by different mechanisms of action.

Laminin is a major component of basement membranes. We previously reported that the globular region of laminin B chain(s) and the cross region of the A chain play an active role in mouse lung branching morphogenesis. In this study, basic morphogenic cell behaviors modulated by laminin were analyzed in order to elucidate how this glycoprotein promotes lung development. Cocultures of epithelial and mesenchymal cells from mouse fetal lungs were used to determine the effect of site-specific monoclonal antibodies to laminin (AL-1, AL-2, AL-3, AL-4, and AL-5) on epithelial and mesenchymal cell adhesion, proliferation, and organotypic rearrangement. We found that monoclonal antibody AL-1, directed against the cross region of the laminin A chain, inhibited epithelial and mesenchymal cell attachment and had a selective antiproliferative effect on epithelial cells. In contrast, monoclonal antibody AL-5, directed against the globular region of the B chain(s), blocked epithelial cell polarity. Immunohistochemical studies on epithelial-mesenchymal cocultures exposed to monoclonal antibody AL-5 revealed the absence of laminin deposition at the epithelial-mesenchymal interface, whereas type collagen IV was present at this site. These findings suggest that each of the two laminin domains involved in lung development promotes morphogenesis by a different mechanism of action. The cross-region of the A chain mediates cell adhesion and epithelial cell proliferation, whereas the globular region of the laminin B chain(s) is critical for the process of basement membrane assembly and cell polarization. The combined effect of both laminin domains on epithelial and mesenchymal cells and on the interaction between them seems to be essential for normal lung branching morphogenesis.

Retinoic acid stimulates mouse lung development by a mechanism involving epithelial-mesenchymal interaction and regulation of epidermal growth factor receptors.

Retinoic acid (RA) stimulated proliferation of both epithelial and mesenchymal cells in cocultures isolated from developing mouse lungs. There was a corresponding increase in epithelial branching activity in organ culture of embryonic lungs exposed to similar doses of RA. Stimulation was maximal with concentrations of 1 microM and progressively decreased with either lower or higher concentrations. However, when lung cell monocultures of isolated epithelial and mesenchymal cells were exposed to RA, the mitogenic effect was observed only in the mesenchymal population. This suggests that RA may not have a direct mitogenic effect on epithelial cells but rather functions indirectly through the mesenchyme. The cellular response to RA was correlated with an increase in the expression of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) also stimulated terminal branch formation in the developing lung. Unlike RA, EGF stimulated proliferation in both epithelial cells and mesenchymal cells in monoculture. In comparison, transforming growth factor-alpha, which also binds to the EGFR, elicited no response. We conclude that RA stimulates cell proliferation and branching activity in the developing mouse lung by a mechanism involving epithelial-mesenchymal interactions. The effect is, in part, produced by stimulation of EGFR expression, with the resulting amplification of the cellular response to EGF or other EGFR ligands. In this process the mesenchyme provides a paracrine support to the epithelium, otherwise unresponsive to RA. Further studies identified the mesenchyme as a major source of EGF in the embryonic lung, suggesting that mesenchymal EGF may represent a paracrine factor involved in the epithelial response to RA.

Laminin expression in the mouse lung increases with development and stimulates spontaneous organotypic rearrangement of mixed lung cells.

The recent establishment of a role for laminin in mouse lung organogenesis (Schuger et al. 1990a,b, 1991) prompted us to study its expression in the developing lung. Laminin A and B chains were detected in the murine lung from the first hours of development onward. In situ hybridization of mRNA as well as SDS-PAGE studies of lung cells in monoculture indicated that both epithelium and mesenchyme produce complete laminin molecules. Quantitative analysis of the in situ hybridization studies showed a gradual increase in laminin expression during development which was further supported by immunohistochemistry and ELISA. The overall pattern of expression suggested that the effects of laminin in morphogenesis were not restricted to a particular stage of development. Furthermore, the increase in expression during late development supported a role for the molecule in the fetal lung, which was not previously established. We next determined whether the increase in laminin production modulated the behavior of fetal lung cells as compared with their embryonic counterparts. We previously showed that organotypic pattern formation does not occur in cultures of mixed embryonic lung cells unless exogenous laminin is added (Schuger et al., 1990b). Organotypic pattern formation is the result of cell sorting into epithelial and mesenchymal compartments and further rearrangement in a pattern resembling the tissue of origin. In the present study, we demonstrated that organotypic pattern formation occurs spontaneously in cultures of mixed fetal lung cells, which express high laminin levels. Pattern formation was abolished by antibodies to laminin. These studies suggest a correlation between laminin expression and the ability of lung cells in culture to reproduce normal tissue patterns. We conclude that laminin is critical for epithelial-mesenchymal recognition and further morphogenic interaction during both the embryonic and fetal stages of lung development.

Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3.

The 7.1-kilobase-pair (kbp) plasmid pFA3 specifies TEM beta-lactamase production in Neisseria gonorrhoeae. We studied the minimal region required for replication of this plasmid in Escherichia coli by constructing a set of nested deletions of the 3.4-kbp PstI-HindIII fragment. The smallest fragment capable of maintenance in E. coli when ligated to a streptomycin-spectinomycin resistance cassette was 2.0 kbp in size and was different from another autonomously replicating fragment of pFA3 reported by K. H. Yeung and J. Dillon (Plasmid 20:232-240, 1988). The fragment contained single BamHI and XbaI sites and specified a 39-K protein. Fragments subcloned from the minimal region or constructed by deletion from the 3' or 5' ends were not capable of autonomous replication. Mutants constructed by end filling and religating DNA cleaved at the BamHI or XbaI sites were not capable of autonomous replication and no longer produced the 39K protein. These results suggest that replication is dependent on the 39K protein. DNA sequence analysis of the region showed an A-T-rich region followed by four 22-bp direct repeats followed by an open reading frame encoding a 39K basic protein.

Plasmid mediated antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae.

The genetic basis of antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae was studied. Two Ontario isolates of A. pleuropneumoniae were found to be resistant to sulfonamides (Su), streptomycin (Sm) and ampicillin (Amp). Resistance to Su and Sm was specified by a 2.3 megadalton (Mdal) plasmid which appeared to be identical to pVM104, which has been described in isolates of A. pleuropneumoniae from South Dakota. Southern hybridization showed that the 2.3 Mdal Su Sm plasmid was highly related to those Hinc II fragments of RSF1010 known to carry the Su Sm genes, but was unrelated to the remainder of this Salmonella resistance plasmid. Resistance to Su and Amp was specified by a 3.5 Mdal plasmid and appeared identical to pVM105 previously reported. The beta-lactamase enzyme had an isoelectric point of approximately 9.0. Southern hybridization showed no relationship to the TEM beta-lactamase. A third isolate of A. pleuropneumoniae was found to be resistant to chloramphenicol (Cm), Su and Sm by virtue of a 3.0 Mdal plasmid which specified a chloramphenicol acetyl transferase. We conclude that resistance to Su, Sm, Amp and Cm is mediated by small plasmids in A. pleuropneumoniae. Although the Su and Sm resistance determinants are highly related to those found in Enterobacteriaceae, the plasmids themselves and the beta-lactamase determinant are different.

Comparative virulence of porcine Haemophilus bacteria.

The virulence of strains of Haemophilus pleuropneumoniae serotype 1, 2, 3, 7 and strains of the "minor-group" and Haemophilus parasuis were compared by inoculating specific pathogen-free pigs into the lower airways with specified doses of bacteria. Haemophilus pleuropneumoniae, strain W, serotype 1, given in 1 X 10(8) colony-forming units, produced a lethal acute pleuropneumonia in four pigs. Nonlethal localized pulmonary necrosis was induced in four groups of two pigs given 1 X 10(7), 1 X 10(6), 1 X 10(5) and 1 X 10(4) respectively of the same strain. Two groups of four pigs developed chronic lesions when inoculated with 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain Shope 4074, serotype 1 and 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain WF83, serotype 7, respectively. Of 20 pigs given 1 X 10(8) colony-forming units of strain 1536, serotype 2, two died of acute pleuropneumonia and 18 had lesions of pulmonary necrosis or abscessation and pleuritis. A dose of 4 X 10(9) colony-forming units of strain BC181, serotype 3, induced pulmonary necrosis similar to the lesions in pigs given 10(7) colony-forming units or less of strain W, serotype 1, suggesting that the serotype 3 strain is less virulent. No clinical signs, but focal areas of pulmonary fibrosis and pleural adhesions were induced in four pigs inoculated with 4 X 10(9) colony-forming units of the "minor-group" strain 7ATS. Similarly, four pigs inoculated with "minor-group" strain 33PN did not show clinical signs, but had focal necrotic and fibrotic pulmonary lesions and pleural adhesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial susceptibility of 51 strains of Haemophilus pleuropneumoniae.

Fifty-one strains of Haemophilus pleuropneumoniae were tested for susceptibility to 27 antimicrobial agents using agar disc diffusion, broth-tube dilution and microdilution methods. There was generally good agreement between the interpretation of the disc diffusion inhibition zones and the actual minimal inhibitory concentrations obtained with the dilution methods. The agreement between the results obtained with the broth-tube dilution method and the microdilution method was very good. Three strains were resistant to penicillin, ampicillin, carbenicillin, methicillin and tetracycline. One of those was also resistant to chloramphenicol. Forty strains were resistant to streptomycin, 23 strains were resistant to novobiocin and seven were resistant to triple sulfa. It is thus necessary to consider resistance development against antimicrobial agents chosen for the treatment of pleuro-pneumonia in pigs caused by Haemophilus pleuropneumoniae.

Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)