The gene expression of environmental organisms is useful as a biomarker of environmental pollution. One of its advantages is high sensitivity. We identified the cDNA of a novel cadmium-responsive gene in the soil collembolan Folsomia candida. The deduced protein, designated “metallothionein-like motif containing protein” (MTC), was cysteine-rich and contained a metallothionein-like motif with similarity to metallothionein, but had a much longer sequence than metallothionein and contained repeated sequences of amino acids. Expression of MTC mRNA was sensitively induced by cadmium exposure at 0.3 mg/kg of dry food, a concentration at which toxic effects are not observed, but expression was not affected by γ-ray exposure (an inducer of oxidative stress). These findings suggest that MTC is involved in cadmium-binding processes rather than in oxidative-stress responses. In conclusion, we suggest that gene expression of MTC may be a candidate biomarker for detecting low levels of cadmium contamination in soil.

The spreading of extended-spectrum β-lactamases (ESBL)-producing thermotolerant coliforms (TC) in the water environment is a threat to human health but little is known about ESBL-producing TCs in the Yangtze River. We received 319 ESBL-producing stains obtained from the Chongqing basin and we investigated antibiotic susceptibility, bla gene types and the presence of integrons and gene cassettes. 16.8% of TC isolates were ESBL-producing bacteria and blaTEM+CTx-M was the predominant ESBL type. 65.2% of isolates contained class 1 integrons, but only 3 carried intI 2. Gene cassettes were amplified and sequenced. aadA, drfA, cmlA, sat1, aar3 and two ORF cassettes were found. In conclusion, Yangtze River is heavily polluted by ESBL-producing TC bacteria and the combined bla gene type could enhance antibiotic resistance. Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance. Characterization of gene cassettes could reveal the dissemination of antibiotic resistance genes.

We evaluated the molecular diversity of narG gene from Suquía River sediments to assess the impact of the nitrate concentration and water quality on the composition and structure of the nitrate-reducing bacterial community. To this aim, a library of one of the six monitoring stations corresponding to the highest nitrate concentration was constructed and 118 narG clones were screened. Nucleotide sequences were associated to narG gene from alpha-, beta-, delta-, gammaproteobacteria and Thermus thermophilus. Remarkably, 18% of clones contained narG genes with less than 69% similarity to narG sequences available in databases. Thus, indicating the presence of nitrate-reducing bacteria with novel narG genes, which were quantified by real-time PCR. Results show a variable number of narG copies, ranging from less than 1.0 × 102 to 5.0 × 104 copies per ng of DNA, which were associated with a decreased water quality index monitored along the basin at different times.

This study investigated antibiotic resistance profiles and tetracycline resistance genes in Enterobacteriaceae family isolates from the Pearl rivers. The Enterobacteriaceae isolates were tested for susceptibility to seven antibiotics ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, sulphamethoxazole/trimethoprim, tetracycline and trimethoprim. In Liuxi reservoir, with an exception to ampicillin resistant strains (11%) no other antibiotic resistance bacterial strains were detected. However, multiple drug resistance in bacterial isolates from the other sites of Pearl rivers was observed which is possibly due to sewage discharge and input from other anthropogenic sources along the rivers. Four tetracycline resistance genes tet A, tet B, tet C and tet D were detected in the isolates from the rivers. The genes tet A and tet B were widely detected with the detection frequencies of 43% and 40% respectively. Ciprofloxacin and levofloxacin resistant enteric bacteria were also isolated from the pig and duck manures which suggest a wider distribution of human specific drugs in the environment. This investigation provided a baseline data on antibiotic resistance profiles and tetracycline resistance genes in the Pearl rivers delta.

The attenuation and fate of erythromycin-resistance-methylase (erm) and extended-spectrum beta-lactamse (bla) genes were quantified over time in aquatic systems by adding 20-L swine waste to 11,300-L outdoor mesocosms that simulated receiving water conditions below intensive agricultural operations. The units were prepared with two different light-exposure scenarios and included artificial substrates to assess gene movement into biofilms. Of eleven genes tested, only erm(B), erm(F), blaSHV and blaTEM were found in sufficient quantity for monitoring. The genes disappeared rapidly from the water column and first-order water-column disappearance coefficients were calculated. However, detected gene levels became elevated in the biofilms within 2 days, but then disappeared over time. Differences were observed between sunlight and dark treatments and among individual genes, suggesting that ecological and gene-specific factors play roles in the fate of these genes after release into the environment. Ultimately, this information will aid in generating better predictive models for gene fate.

The responses of Dicentrarchus labrax and Liza aurata to aquatic pollution were assessed in a contaminated coastal lagoon, using both traditional and novel biomarkers combined. DNA damage, assessed by comet assay, was higher in both fish species from the contaminated sites, whereas levels of cytochrome P450 1A1 gene expression were not significantly altered. The liver histopathological analysis also revealed significant lesions in fish from contaminated sites. Alterations in ras and xpf genes were analysed and additional pollutant-responsive genes were identified. While no alterations were found in ras gene, a downregulation of xpf gene was observed in D. labrax from a contaminated site. Suppression subtractive hybridization applied to D. labrax collected at a contaminated site, revealed altered expression in genes involved in energy metabolism, immune system activity and antioxidant response. The approach and results reported herein demonstrate the utility of anchoring traditional biomarker responses alongside novel biomarker responses.

Background, aim, and scope Pharmaceuticals are emerging pollutants widely used in everyday urban activities which can be detected in surface, ground, and drinking waters. Their presence is derived from consumption of medicines, disposal of expired medications, release of treated and untreated urban effluents, and from the pharmaceutical industry. Their growing use has become an alarming environmental problem which potentially will become dangerous in the future. However, there is still a lack of knowledge about long-term effects in non-target organisms as well as for human health. Toxicity testing has indicated a relatively low acute toxicity to fish species, but no information is available on possible sublethal effects. This study provides data on the physiological pathways involved in the exposure of Atlantic salmon as representative test species to three pharmaceutical compounds found in ground, surface, and drinking waters based on the evaluation of the xenobiotic-induced impairment resulting in the activation and silencing of specific genes. Materials and methods Individuals of Atlantic salmon (Salmo salar) parr were exposed during 5 days to environmentally relevant concentrations of three representative pharmaceutical compounds with high consumption rates: the analgesic acetaminophen (54.77 ± 34.67 µg L⁻¹), the anticonvulsant carbamazepine (7.85 ± 0.13 µg L⁻¹), and the beta-blocker atenolol (11.08 ± 7.98 µg L⁻¹). Five immature males were selected for transcriptome analysis in brain tissues by means of a 17k salmon cDNA microarray. For this purpose, mRNA was isolated and reverse-transcribed into cDNA which was labeled with fluorescent dyes and hybridized against a common pool to the arrays. Lists of significantly up- and down-regulated candidate genes were submitted to KEGG (Kyoto Encyclopedia of Genes and Genomes) in order to analyze for induced pathways and to evaluate the usefulness of this method in cases of not completely annotated test organisms. Results Exposure during 5 days to environmentally relevant concentrations of the selected pharmaceutical compounds acetaminophen, carbamazepine, and atenolol produced differences in the expression of 659, 700, and 480 candidate genes, respectively. KEGG annotation numbers (KO annotations) were obtained for between 26.57% and 33.33% of these differently expressed genes per treatment in comparison to non-exposure conditions. Pathways that showed to be induced did not always follow previously reported targets or metabolic routes for the employed treatments; however, several other pathways have been found (four or more features) to be significantly induced. Discussion Energy-related pathways have been altered under exposure in all the selected treatments, indicating a possible energy budget leakage due to additional processes resulting from the exposure to environmental contaminants. Observed induction of pathways may indicate additional processes involved in the mode of action of the selected pharmaceuticals which may not have been detected with conventional methods like quantitative PCR in which only suspected features are analyzed punctually for effects. The employment of novel high-throughput screening techniques in combination with global pathway analysis methods, even if the organism is not completely annotated, allows the examination of a much broader range of candidates for potential effects of exposure at the gene level. Conclusions The continuously growing number of annotations of representative species relevant for environmental quality testing is facilitating pathway analysis processes for not completely annotated organisms. KEGG has shown to be a useful tool for the analysis of induced pathways from data generated by microarray techniques with the selected pharmaceutical contaminants acetaminophen, carbamazepine, and atenolol, but further studies have to be carried out in order to determine if a similar expression pattern in terms of fold change quantity and pathways is observed after long-term exposure. Together with the information obtained in this study, it will then be possible to evaluate the potential risk that the continuous release of these compounds may have on the environment and ecosystem functioning.

Purpose Experiments were conducted to examine the effects of mercury (Hg) on soil nitrification activities and the microbial communities of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Methods The soil samples spiked with different Hg concentrations were incubated for a period of 1, 2, 4, and 8 weeks in triplicate and the potential nitrification rate (PNR) of the samples was determined. The abundance of AOB and AOA was measured after an 8-week incubation by real-time polymerase chain reaction (PCR) assay of the amoA genes, while the community compositions by cloning and sequencing approaches. Results The soil PNR differed with different incubation periods. It tended to decrease with increasing soil Hg concentrations at week 1, basing on which the half-maximal effective concentration (EC50) was 1.59 mg kg⁻¹. There was no significant difference in the abundance of AOB or AOA among the treatments. The AOB community was dominated by Nitrosospira-like sequences and more than 70% of the obtained clones were affiliated with the cluster 3a.2. The percentage of cluster 3a.1 in AOB community appeared to a consistent trend of decreasing with ascending soil Hg concentrations. While all the AOA sequences in the clone libraries were grouped into cluster S (soil and sediment origin). Conclusions This study revealed that Hg could inhibit soil potential nitrification and the extent varied with incubation periods. Soil Hg pollution changed the composition of soil AOB to some extent. These findings will be helpful to recognize the effects of Hg on the activity and community composition of soil ammonia oxidizers.

Background, aim, and scope Current Australian legislation permits the beneficial application of grease trap waste (GTW) to agricultural soil, viewing it as a beneficial source of organic matter and soil conditioner containing no/low amounts of metals or pathogenic organisms. However, little is known about the influence of GTW on soil bacterial community. A field experiment was established at Menangle in south western Sydney in Australia to quantitatively assess the impacts of different types (GTW CO and GTW CL) and amounts of GTW application on the soil bacterial community and diversity. Furthermore, a municipal solid waste (MSW) compost was simultaneously examined to compare against the other organic wastes. Knowledge about the shifts in microbial community structure and diversity following the applications of organic wastes could help to evaluate the ecological consequences on the soil and thus to develop sound regulatory guidelines for the beneficial reuse of organic wastes in agricultural lands. Materials and methods Soil samples were collected from recycled organics plots treated with different types and quantity of organic wastes. The field experimental treatments included control (CK, without application of any organic wastes), low amount of GTW CO (COL), GTW CL (CLL), and MSW (ML), and high amounts of GTW CO (COH), GTW CL (CLH), and MSW compost (MH). Microbial DNA was extracted from soil samples and the 16S rRNA genes were polymerase chain reaction (PCR)-amplified. The PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The bacterial community structures and diversity were assessed using the DGGE profiles and clone libraries constructed from the excised DGGE bands. Results DGGE-based analyses showed that application of the GTW CO, regardless of the amount applied, had significant negative effects on soil bacterial genotypic diversity and community structure compared with the control, while the applications of other organic wastes including the GTW CL and MSW had no clear effects. The effects of the rate of organic waste application on soil bacterial community characteristics varied with the types of organic wastes applied. Sequence-based analyses of 126 clones indicated that Proteobacteria (53.2%) was the dominant taxa at the experimental site, followed by Actinobacteria (9.5%), Bacteroidetes (7.9%), Firmicutes (7.9%), Gemmatimonadetes (5.6%), Chloroflexi (2.4%), Acidobacteria (1.6%) and the unclassified group (11.9%). In the COH treatment, Acidobacteria, Bacteroidetes, and Gemmatimonadetes were not detected; the percentages of Firmicutes, Proteobacteria, and Actinobacteria in the COH treatment were significantly different from those in CK. There is a significant positive correlation (r = 0.71, p = 0.002) between the C/N ratio of organic wastes and the bacterial genotypic communities. Discussion Both the type and the amount of GTW applied affected soil bacterial genotypic diversity and community structure. The different effects of various types of organic wastes on soil bacterial characteristics may be predicted by the differences in specific properties of organic wastes such as C/N ratio, as evidenced by the strong and significant positive relationship between the bacterial community distance and the environmental distance of C/N ratio. This also indicates that the C/N ratio of GTW applied can be a major driver for the shift in the soil bacterial community. Conclusions Our results revealed that the effects of organic wastes on soil bacterial communities varied with the types of organic wastes, and depending on the rate of application. Application of the GTW CO led to significant shifts in soil bacterial community diversity and structure. The effects of different types of organic wastes on the soil bacterial characteristics can be predicted by the differences of specific properties of organic wastes, such as the C/N ratio. Sequence-based analyses of 126 clones indicated that Proteobacteria was the dominant taxa at the experimental site. Recommendations and perspectives Our results have important implications for developing sound regulatory guidelines for the beneficial reuse of organic wastes, indicating that GTW CO and similar organic waste treatments may not be suitable for application in agricultural soils due to its significant negative effect on soil bacterial community.

Background, aim, and scope Linear alkylbenzene sulfonate (LAS) is the most used anionic surfactant in a worldwide scale and is considered a high-priority pollutant. LAS is regarded as a readily biodegradable product under aerobic conditions in aqueous media and is mostly removed in wastewater treatment plants, but an important fraction (20-25%) is immobilized in sewage sludge and persists under anoxic conditions. Due to the application of the sludge as a fertilizer, LAS reaches agricultural soil, and therefore, microbial toxicity tests have been widely used to evaluate the influence of LAS on soil microbial ecology. However, molecular-based community-level analyses have been seldom applied in studies regarding the effects of LAS on natural or engineered systems, and, to our knowledge, there are no reports of their use for such appraisals in agricultural soil. In this study, a microcosm system is used to evaluate the effects of a commercial mixture of LAS on the community structure of Alphaproteobacteria, Actinobacteria, and Acidobacteria in an agricultural soil. Material and methods The microcosms consisted of agricultural soil columns (800 g) fed with sterile water (8 ml h⁻¹) added of different concentration of LAS (10 or 50 mg l⁻¹) for periods of time up to 21 days. Sterile water was added to control columns for comparison. The structures of Alphaproteobacteria, Actinobacteria, and Acidobacteria communities were analyzed by a cultivation independent method (temperature gradient gel electrophoresis (TGGE) separation of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes). Relevant populations were identified by subsequent reamplification, DNA sequencing, and database comparisons. Results Cluster analysis of the TGGE fingerprints taking into consideration both the number of bands and their relative intensities revealed that the structure of the Alphaproteobacteria community was significantly changed in the presence of LAS, at both concentrations tested. The average number of bands was significantly lower in the microcosms receiving 50 mg l⁻¹ LAS and in the lower portion of soil cores. The clear differentiation of the samples of the upper portion of the soil columns amended with LAS was specifically related to the presence and intensity of a distinctive major band (named band class 7). There was a statistically significant positive correlation between the concentrations of LAS detected in soil portions taken from LAS 10 mg l⁻¹ and LAS 50 mg l⁻¹ microcosms and the relative intensity of band class 7 in the corresponding TGGE profiles. Prevalent Alphaproteobacteria populations in the soil microcosms had close similarity (>99%) to cultivated species affiliated to genera of the Rhizobiaceae, Methylocystaceae, Hyphomicrobiaceae, Rhodospirillaceae, Brucellaceae, Bradyrhizobiaceae, and Caulobacteraceae families. The population represented by band class 7 was found closely related to the genus Phenylobacterium (Caulobacteraceae). According to cluster analysis of TGGE profiles, the structure of both Actinobacteria and Acidobacteria communities in the soil microcosms was remarkably stable in the presence of LAS at the two concentrations tested, as most bands were universally present in all samples and displayed fairly similar relative intensities. Discussion Previous studies by others authors, based on biological and chemical tests, concluded that LAS toxicity was not an important microbial selection factor in sludge amended soil, while work based on the use of molecular fingerprinting to evaluate the impact of LAS in aqueous media and marine sediments showed that concentrations as low as 1 mg l⁻¹ significantly influence the development of the bacterial community structure. Although TGGE is not a strictly quantitative method due to the bias introduced by the PCR reaction, changes of band intensity through experiments are a consequence of a change in the relative abundance of the corresponding populations in the community and can be used as a semiquantitative measure of bacterial diversity. Our results evidence that the Phenylobacterium population represented by band class 7 was favored by the presence of increasing concentrations of LAS in the soil and turned into a dominant population, suggesting its possible ability to use LAS in soil as a source of nutrients. As studies with pure cultures are required to confirm the ability of this population to degrade LAS, isolation strategies are currently under development in our laboratory. The weak effect of LAS on the structure of Actinobacteria and Acidobacteria communities is particularly interesting, as to our knowledge, there are no previous reports regarding the effects of LAS on these bacterial groups in soil. Conclusions, recommendations, and perspectives The Phenylobacterium-related alphaproteobacterial population identified in this work was selectively enriched in LAS polluted soil and is a plausible candidate to play a relevant role in the biotransformation of the surfactant under the conditions tested. The surfactant had no remarkable effects on the Actinobacteria and Acidobacteria fingerprints in soil, even when present at concentrations widely exceeding those reached in soil immediately after sludge application. TGGE fingerprinting provides a reliable and low time-consuming method for the monitoring of the bacterial community structure and dynamics, and we recommend its integration with the biological and chemical analyses usually applied in risk assessment of LAS in the environment.