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.

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.

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)