Detection of sulfonamide resistance genes via in situ PCR-FISH.

Due to the rising use of antibiotics and as a consequence of their concentration in the environment an increasing number of antibiotic resistant bacteria is observed. The phenomenon has a hazardous impact on human and animal life. Sulfamethoxazole is one of the sulfonamides commonly detected in surface waters and soil. The aim of the study was to detect sulfamethoxazole resistance genes in activated sludge biocenosis by use of in situ PCR and/or hybridization. So far no FISH probes for the detection of SMX resistance genes have been described in the literature. We have tested common PCR primers used for SMX resistance genes detection as FISH probes as well as a combination of in situ PCR and FISH. Despite the presence of SMX resistance genes in activated sludge confirmed via traditional PCR, the detection of the genes via microscopic visualization failed.

Resistance of Escherichia coli and Enterococcus spp. to selected antimicrobial agents present in municipal wastewater.

In this study, the susceptibility to erythromycin (E) and to trimethoprim/sulfamethoxazole (SXT) among isolates of Enterococcus spp. and Escherichia coli was tested, respectively. Both fecal indicators were detected and isolated from raw (RW) and treated wastewater (TW) as well as from samples of activated sludge (AS) collected in a local wastewater treatment plant (WWTP). Biodiversity of bacterial community in AS was also monitored using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Additionally, distribution of sul1-3 genes conferring sulfonamide resistance was tested among SXT-resistant E. coli. Simultaneously, basic physicochemical parameters and concentrations of eight antimicrobial compounds (belonging to folate pathway inhibitors and macrolides class) were analyzed in RW and TW samples. Six of the selected antimicrobial agents, namely: erythromycin, clarithromycin, trimethoprim, roxithromycin, sulfamethoxazole, and N-acetyl-sulfamethoxazole were detected in the wastewater samples. Bacterial biodiversity of AS samples were comparable with no relevant differences. Among tested Enterococcus spp., E-resistant isolates constituted 41%. SXT resistance was less prevalent in E. coli with 11% of isolates. The genes conferring resistance to sulfonamides (sul1-3) were detected in SXT-resistant E. coli of wastewater origin with similar frequencies as in other environmental compartments, including clinical ones.

Removal of petroleum pollutants and monitoring of bacterial community structure in a membrane bioreactor.

The long-term operational stability (159 d) in removal of organics and ammonia from synthetic wastewater was investigated. The experiment was carried out in two identical plug flow membrane bioreactors (MBR) (each with a submerged A4 Kubota membrane) operated under aerobic conditions. The vacuum distillate of a crude oil fraction in the emulsified state, which was used to model the petroleum pollutants, was added into the feed medium. The performance of biological treatment was evaluated by physicochemical analyses such as nitrogen forms, COD, and BOD. Additionally, monitoring of PAHs in the wastewaters was performed using HPLC-diode array detector. Moreover, the community structure of bacteria was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis. The MBR treatment was very effective with reduction by more than 90% of COD and Total Organic Carbon. Nearly complete removal of petroleum originated non-polar micropollutants was observed. The influence of the highest dosage of petroleum pollutants (1000 µLL(-1)) on the bacterial community was noted.

Ammonia oxidizing bacteria community in activated sludge monitored by denaturing gradient gel electrophoresis (DGGE).

Biological treatment based on activated sludge is efficient in municipal wastewater treatment. The removal of nitrogen compounds is crucial for sewage purification. Due to the fact that ammonia is toxic for a water environment and causes eutrophication, ammonia oxidation is of the utmost importance in wastewater treatment. Observing the changeability of ammonia oxidizing bacteria (AOB) and identifying their most abundant species can be helpful in the optimization of wastewater treatment. In this study we used denaturing gradient gel electrophoresis (DGGE), combined with cloning and sequencing of 16S rRNA and AmoA gene fragments in order to estimate AOB biodiversity and temporal community changes. Activated sludge samples were collected from the municipal WWTP in Gliwice (Poland) at 2-week intervals. Ammonia concentration in the influent during the experiment was 30.2-57.6 mg N-NH4+/L. The research revealed a high diversity of uncultured bacteria. It is suspected that these bacteria could be involved in the nitrification, which points to the fact that these bacteria might be efficient in the process. However such a situation is not confirmed and it requires further research. The appearance of Ferribacterium-like bacteria together with Nitrosomonas sp. as the most abundant bacteria was found.

Molecular analysis of temporal changes of a bacterial community structure in activated sludge using denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization (FISH).

Wastewater treatment based on activated sludge is known to be one of the most effective and popular wastewater purification methods. An estimation of microbial community variability in activated sludge allows us to observe the correlation between a particular bacterial group's appearance and the effectiveness of the removal of chemical substances. This research is focused on microbial community temporal changes in membrane bioreactors treating wastes containing a high level of ammonia nitrogen. Samples for this study were collected from two membrane bioreactors with an activated sludge age of 12 and 32 days, respectively. The activated sludge microbial community was adapted for the removal of ammonia nitrogen up to a level of 0.3 g NH4(+) - N g/VSS/d (VSS - volatile suspended solids). The methods - denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA gene PCR products and fluorescent in situ hybridization (FISH) with 16S rRNA gene probes - revealed significant differences in the microbial community structure in the two bioreactors, caused mainly by a difference in sludge age. According to the results obtained in this study, a bioreactor with a sludge age of 12 days is characterized by a much higher microbial community diversity than a bioreactor with a sludge age of 32 days. Interestingly, the appearance of particular species of nitrifying bacteria was constant throughout the experiment in both bioreactors. Changes occured only in the case of the Nitrosomonas oligotropha lineage bacteria. This study demonstrates that the bacterial community of bioreactors operating with different sludge ages differs in total community structure. Nevertheless, the changeability of the bacterial community structure did not have any influence on the efficiency of nitrification.