The microbiology of a pharmaceutical effluent and its public health implications

The effluent of a pharmaceutical company was examined microbiologically. Its bacterial count was 2.15 × 10⁵ c.f.u./ml and there was evidence of faecal contamination with MPN of > 1800. The organisms encountered included Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa. The resistances of the 25 bacterial strains isolated from the effluent to the commonly used antibiotics were studied. About 80% of the isolates were resistant to Amoxycillin, 76% to Nitrofurantoin, 64% to Cotrimoxazole and Augmentin, 60% were resistant to Nalidixic acid, 52% were resistant to Tetracycline and Ofloxacin, while resistance of 12% was obtained for Gentamicin. Among the eight antibiotics tested, seven patterns of drug resistance were obtained and all of them were multiple-drug resistance with the number of antibiotics ranging from 2–8. All the strains of E. coli and S. aureus had high MIC values for Cloxacillin and Amoxycillin. In all, 13 strains of the bacterial isolates had evidence for the production of -lactamases. The potential of the effluent in spreading drug resistance and the public health implications are discussed.     

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

Mesophilic and thermophilic activated sludge post treatment of anaerobic effluent. Sludge and wastewater characterisation using batch experiments

Anaerobic pretreated paper process water was characterized interms of readily biodegradable, slowly biodegradable, very slowly biodegradable and inert wastewaterfractions under mesophilic and thermophilic conditions. The anaerobic pretreated paper process water containeda relatively high amount of slowly biodegradable components and few easily biodegradable componentsas indicated by the ratio of short term BOD over the BOD5. Wastewater readily biodegradable COD, determinedas short term BOD, was almost similar when measured under both temperature conditions. Fractions ofslowly biodegradable COD and inert COD of the same wastewater were found to depend on the type of biomassinvolved in the test. Thermophilic aerobic biomass was not able to degrade the wastewater to the sameextent as the mesophilic biomass resulting in higher apparent inert COD levels. Furthermore, wastewater colloidalCOD did not flocculate under thermophilic conditions and was thus not removed from the liquid phase.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

Reduced byssal thread production and movement by the intertidal mussel<Emphasis Type="Italic"> Hormomya mutabilis</Emphasis> in response to effluent from predators

Laboratory experiments were carried out to investigate byssal thread production by the intertidal mytilid mussel Hormomya mutabilis in response to effluent from the predatory crab Eriphia smithii and the starfish Coscinasterias acutispina. During the early period of the experiment, large H. mutabilis exposed to crab effluent produced a significantly smaller number of functional byssal threads than mussels in crab-free water. No significant difference in the diameter of threads produced in the two treatments was detected. The number of functional byssal threads produced by small H. mutabilis exposed to crab effluent did not differ significantly from that of mussels in crab-free water. However, small H. mutabilis exposed to crab effluent tended to discard fewer byssal bundles, that is, they shifted their attaching sites less frequently than similar mussels in crab-free water. In the presence of waterborne cues from the crab, H. mutabilis tended to reduce both the secretion of byssal threads and movement across the substratum. No significant differences in behaviour were observed between large mussels exposed to effluent from the starfish and those unexposed. The adaptive significance of the responses shown by H. mutabilis is discussed in terms of protection against predators differing in foraging behaviour. Electronic Publication     

Factors affecting reproductive performance in Ontario dairy herds

Data were collected on reproductive, health and production events in 45 dairy herds in southwestern and eastern Ontario, Canada, from July 1990 to July 1993. Mean and median days in milk at first estrus, at first service, and at conception were 85.1 and 83.9, 94.0 and 88.9, and 126.3 and 115.5 d, respectively. The mean estrus detection rate was 48.3%, with a minimum of 28.4% and a maximum of 64.2%. The mean overall conception rate was 46.7%, with a minimum of 29.8% and a maximum of 70.7%. The most commonly used used therapeutic agents were GnRH (11.5% lactational incidence risk) and PG (13.1% lactational incidence risk). Estrus management accounted for the largest portion of PG use in the herds studied (69.4%). Results of a two sample t-test indicated no significant difference in reproductive performance among herds housed in free-stall compared with those housed in tie-stall barns. Multivariate linear regression models of 3 reproductive performance measures indicated that cows with ovarian cysts and/or increased peak milk production had significantly longer calving-to-first estrus and calving-to-conception intervals as well as lower conception rates than average producing cows without cysts. Cows with metritis were found to have significantly shorter calving-to-conception intervals but no detectable difference in conception rate or calving-to-first estrus interval than their herdmates. As lactation number increased reproductive performance improved. Cows that were observed in estrus earlier conceived earlier. Animals receiving uterine infusions had significantly longer calving-to-conception intervals and lower conception rates than their herdmates. There was no detectable difference in reproductive performance for cows treated with GnRH or PG compared with the nontreated cows. An increased estrus detection rate was associated with shorter calving-to-conception intervals and lower conception rates. Based on the range of performance in the study herds, the factors with the greatest potential influence on calving-to-conception interval in the average herd were the estrus detection rate and conception rate.     

Characterization of Shiga toxin‐producing Escherichia coli isolated from dairy cows in Argentina

Aims: To feno‐genotypically characterize the Shiga toxin‐producing Escherichia coli (STEC) population in Argentinean dairy cows. Methods and Results: From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52%stx2 and 37%stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty‐five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29%stx2, saa, ehxA. One hundred and fifty‐six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2⁺, eae⁺, ehxA⁺ virulence pattern. Conclusions: We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. Significance and Impact of the study: The non‐O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.     

A combined immunomagnetic separation and lateral flow method for a sensitive on-site detection of Bacillus anthracis spores – assessment in water and dairy products

Aim:  Combination of immunomagnetic separation (IMS) and lateral flow device (LFD) assays for the development of a sensitive, rapid, on-site methodology that enables concentration and detection of Bacillus anthracis spores in complex samples.
Methods and Results:  The data presents the development of an optimized, 30 min, IMS assay, with about 95% capture of B. anthracis spores from different dairy products ( n  = 38). No cross reactivity was detected with typical milk flora and some closely related Bacilli. To enable direct application of the IMS captured spores on the LFD, spores were eluted from the bead–spore complex utilizing 95% (v/v) formamide-10 mmol l⁻¹ EDTA for 30 s in a microwave oven. Detached spores were analysed on LFD enabling detection within 10 min. The combined IMS–LFD methodology (40 min) demonstrates a 60-fold improvement in sensitivity, relative to samples that were applied directly on the LFD without the IMS concentrating step.
Conclusions:  The IMS–LFD method is a powerful platform, combining rapidity, specificity and efficiency for concentrating and detecting B. anthracis from water and milk contaminated samples.
Significant and Impact of the Study:  The combination of IMS and LFD enhances the sensitivity and flexibility of B. anthracis spore detection from complex samples. This method can potentially be extended to other toxins and micro-organisms in a variety of matrices.     

Effects of an energy-dense diet and nicotinic acid supplementation on production and metabolic variables of primiparous or multiparous cows in periparturient period

It is well observed that feeding energy-dense diets in dairy cows during the dry period can cause metabolic imbalances after parturition. Especially dairy cows with high body condition score (BCS) and fed an energy-dense diet were prone to develop production diseases due to metabolic disturbances postpartum. An experiment was conducted to determine the effects of an energy-dense diet and nicotinic acid (NA) on production and metabolic variables of primiparous and multiparous cows in late pregnancy and early lactation which were not pre-selected for high BCS. Thirty-six multiparous and 20 primiparous German Holstein cows with equal body conditions were fed with energy-dense (60% concentrate/40% roughage mixture; HC group) or adequate (30% concentrate/70% roughage mixture; LC group) diets prepartum. After parturition, concentrate proportion was dropped to 30% for all HC and LC groups and was increased to 50% within 16 days for LC and within 24 days for HC cows. In addition, half of the cows per group received 24 g NA supplement per day and cow aimed to attenuate the lipid mobilisation postpartum. Feeding energy-dense diets to late-pregnant dairy cows elevated the dry matter (p < 0.001) and energy intake (p < 0.001) as well as the energy balance (p < 0.001) without affecting the BCS (p = 0.265) during this period. However, this did not result in any metabolic deviation postpartum as the effects of prepartum concentrate feeding were not carried over into postpartum period. Multiparous cows responded more profoundly to energy-dense feeding prepartum compared with primiparous cows, and parity-related differences in the transition from late pregnancy to lactation were obvious pre- and postpartum. The supplementation with 24 g NA did not reveal any effect on energy metabolism. This study clearly showed that energy-dense feeding prepartum did not result in metabolic imbalances postpartum in multiparous and primiparous cows not selected for high BCS. A genetic predisposition for an anabolic metabolic status as indicated by high BCS may be crucial for developing production diseases at the onset of lactation.