Lucia Birošová

Development of triclosan and antibiotic resistance in Salmonella enterica serovar Typhimurium

The possible association between the use of triclosan and the development of antibiotic resistance was examined in triclosan-resistant mutants of Salmonella enterica serovar Typhimurium. These mutants were obtained from a sensitive parental strain and from ciprofloxacin-resistant isogenic strains using spontaneous mutagenesis or selection after one short exposure or continuous exposure to low concentrations of triclosan. The results showed that triclosan in the environment does not increase the mutation frequency but selects bacterial strains with reduced antibiotic susceptibility. This property depended on the multiple antibiotic resistance (Mar) phenotype of bacterial strains and on the triclosan concentration.

National study of illicit drug use in Slovakia based on wastewater analysis

The aim of this study was to analyze illicit drugs and their metabolites in wastewater from eight selected wastewater treatment plants (WWTPs) in Slovakia. The effect of two of the biggest music festivals in Slovakia on illicit drugs in wastewater was also investigated. Urinary bio-markers of amphetamine, methamphetamine, cocaine, cannabis and ecstasy use were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We then compared our results with data obtained in other parts of Europe and the world. This study demonstrates that Slovakia has one of highest methamphetamine consumption rates in Europe. Within Slovakia, the highest level of methamphetamine consumption was found in Petržalka, where the mean specific load of this drug in sewage was 169 mg/day/1000 inhabitants; the next highest loads were detected in Piešťany (128 mg/day/1000 inhabitants) and Bratislava (124 mg/day/1000 inhabitants). Amphetamine, ecstasy and cannabis consumption in our study were comparable to that found in other European cities, whereas cocaine consumption was lower. We also analyzed the pattern of drug consumption over the course of a week. The load of the cocaine metabolite benzoylecgonine in wastewater increased during the weekend. The use of this drug was most common in the capital of Slovakia. Increased consumption was also found during a folk festival in Piešťany. The ecstasy load in wastewater from larger cities also significantly increased over the weekend. An increase of drug consumption was also detected during a music festival in Trenčín, especially for ecstasy. The specific load of ecstasy during this festival increased from 3mg/day/1000 inhabitants to 29 mg/day/1000 inhabitants. The possible influence of music styles on the consumption of certain drugs was also observed. During a folk festival, methamphetamine and cocaine were more commonly used.

Pilot study of seasonal occurrence and distribution of antibiotics and drug resistant bacteria in wastewater treatment plants in Slovakia.

This work presents environmental and quality-control data from the analyses of 33 antibiotics in influent and effluent water from two waste water treatment plants (WWTPs) in the capital and the biggest city of Slovakia. Seeing that consumption of antibiotics depends on epidemiological season, samples were collected during February and August. Among assessed antibiotics ciprofloxacin and clarithromycin were detected in highest concentrations in influent water. Seasonal changes were observed only in plant A when antibiotic concentrations decreased. On the other hand an increase in some cases was observed in plant B. Insufficient degradation of some macrolides, sulfonamides and trimethoprim was detected according to their higher concentrations in effluent water. Contact of antibiotics in subinhibitory concentrations and sludge bacteria in WWTPs represent the base for the development of significant levels of microbial resistance. Simultaneously, antibiotic resistance of fecal coliforms and fecal streptococci from sewage sludge was evaluated. Majority of coliform bacteria were found to be resistant to ampicillin and gentamicin. A significant seasonal difference was determined only in case of high-level resistance. In summer samples, an increase in the strains resistant to concentrations higher than the resistance breakpoints established by EUCAST and NCCLS was observed. No antibiotic resistance in streptococci was observed. However, as a part of sewage sludge is mixed with compost and utilized in agriculture, better processing of sludge should be considered.

Fenton-like reaction: A possible way to efficiently remove illicit drugs and pharmaceuticals from wastewater

We analyzed 13 psychoactive pharmaceuticals, illicit drugs and their metabolites in wastewater treatment plant influent and effluent and the possibility of their degradation by biological and chemical processes. Tramadol (413-853 ng/L) and methamphetamine (460-682 ng/L) were the most concentrated compounds in the wastewater in winter and summer, respectively. A significant decrease in the concentration of tramadol in wastewater was measured during the summer. The lowest efficiency was observed for tramadol, venlafaxine, citalopram and oxazepam (∼ 10%) and the highest efficiency was observed for amphetamine and THC-COOH (∼ 80%). The efficiency of compound degradation via the Fenton reaction, a modified Fenton reaction and different degradation (by algae, wood-rotting fungi and enzymes at influent versus effluent) was determined. The Fenton reaction and its modification were efficient at eliminating these substances in comparison with the tested biological processes.

Utilization of Fenton-like reaction for antibiotics and resistant bacteria elimination in different parts of WWTP.

Utilization of relatively low-cost modification of Fenton reaction for the elimination of selected antibiotics and resistant coliforms in different part of wastewater treatment plant (WWTP) was studied. The concentration of antibiotics and occurrence of resistant gems in different stages of WWTP in the capital city of Slovakia - Bratislava was analyzed by LC-MS/MS technique. Consequently, Fenton-like reaction was applied for the elimination of chemical and biological contaminants. Comparative study with classical Fenton reaction was also done. Very high concentrations of clarithromycin, ciprofloxacin and azithromycin in influent water were found. Coliform bacteria were predominantly resistant to ampicillin, ciprofloxacin and gentamicin. After the mechanical stage, the concentration of antibiotics in water was significantly decreased because of the sorption during this step. Biological step degraded 12 types of antibiotics. Analyses of effluent water showed very bad elimination of azithromycin (919ng/L) and clarithromycin (684ng/L). Contrary, ciprofloxacin was removed with very high efficiency (95%). The number of resistant bacteria was also significantly decreased in effluent water. In the case of Escherichia coli only ampicillin and gentamicin resistance bacteria were detected. Our results show that antibiotics as well as resistant bacteria were eliminated by the modification of classical Fenton reaction with high efficiency. The modification of the Fenton reaction can decrease the process wages, environmental impact. Moreover, the degradation process was easily controlled, monitored and tuned.

Fate of psychoactive compounds in wastewater treatment plant and the possibility of their degradation using aquatic plants.

In this study we analyzed and characterized 29 psychoactive remedies, illicit drugs and their metabolites in single stages of wastewater treatment plants in the capital city of Slovakia. Psychoactive compounds were present within all stages, and tramadol was detected at a very high concentration (706 ng/L). Significant decreases of codeine, THC-COOH, cocaine and buprenorphine concentration were observed in the biological stage. Consequently, we were interested in the possibility of alternative tertiary post-treatment of effluent water with the following aquatic plants: Cabomba caroliniana, Limnophila sessiliflora, Egeria najas and Iris pseudacorus. The most effective plant for tertiary cleansing was I. pseudacorus which demonstrated the best pharmaceutical removal capacity. After 48 h codeine and citalopram was removed with 87% efficiency. After 96 h were all analyzed compounds were eliminated with efficiencies above 58%.

Zerovalent iron and iron(VI): Effective means for the removal of psychoactive pharmaceuticals and illicit drugs from wastewaters

Herein we report the analysis of 27 selected psychoactive compounds found in the wastewater of the largest suburb in the eastern part of Central Europe Bratislava—Petržalka, Slovakia. Thirteen of them (MDMA, methamphetamine, amphetamine, THC-COOH, benzoylecgonine, codeine, tramadol, venlafaxine, oxazepam, citalopram, methadone, EDDP, cocaine) were found in concentrations above 30 ng/L. These compoundswere selected for further monitoring. The possibility of complete degradation of these 13 substances by zerovalent iron and iron(VI) was studied in thewastewater from the Petržalka treatment plant. During the week the concentration of themajority of the studied compounds inwastewaterwas stable. Concentrations of MDMA, cocaine, tramadol, and oxazepam reached significantly higher levels during the weekend.Only about 10% removal efficiency for tramadol, venlafaxine, oxazepam, MDMA, citalopram, methadone, and EDDP was observed at the treatment plant. In contrast, methamphetamine, amphetamine, and codeine were removed with 68%, 83%, and 53% efficiency, respectively. The degradation of synthetic drugs (methamphetamine, cocaine, MDMA) in wastewater is limited, while cannabis (of natural biological origin) is degradedwith efficiency greater than 90%. After utilization of the Fenton reaction, its modification, and use of ferrate(VI), a high efficiency of eliminating all of these substances to values below the limit of detection was achieved.

Evaluation of different smoking habits during music festivals through wastewater analysis.

Wastewater analysis is a powerful method that can provide useful information about the abuse of legal and illicit drugs. The aim of our study was to determine nicotine consumption during four different music festivals and to find a connection between smoking and preferences for specific music styles using wastewater analysis. The amount of the nicotine metabolite cotinine was monitored in wastewater at the influent of three waste water treatment plants WWTPs in the Czech Republic and Slovakia, where the festivals took place. Urinary bio-markers of nicotine utilization were analyzed by LC-HRMS. More than 80,000 festival participants were monitored during our study from June to September 2014. A significant increase of nicotine consumption was observed in wastewaters during music festivals. The nicotine ingestion level was back-calculated and expressed as mass of pure drug consumed per day and per 1000 inhabitants for selected cities of both countries. The highest differences between typical levels of cotinine in wastewaters and the levels during music festivals were detected in Piešťany: 4 g/L/1000 inhabitants during non-festival days compared to 8 g/L/1000 inhabitants during the Topfest pop-rock festival and 6g/L/1000 inhabitants during the Grape dance festival. No significant increase of the amounts of cotinine in wastewater was recorded for the Country and Folk festivals.

Newly synthesized indolizine derivatives – antimicrobial and antimutagenic properties

A series of indolizine derivatives have been synthesized and subjected to antibacterial screening studies. Antibacterial activity of 21 derivatives was investigated against Staphylococcus aureus, Mycobacterium smegmatis, Salmonella typhimurium and Escherichia coli; also, the sensitivity of model yeast Candida parapsilosis and some model filamentous fungi Aspergillus fumigatus, Alternaria alternata, Botrytis cinerea and Microsporum gypseum was tested. Newly synthesized indolizine derivatives have shown selective toxicity to Gram-positive bacteria S. aureus and were also considered to be able to inhibit the acidoresistant rod M. smegmatis. Derivative XXI has shown the highest inhibition effect with the bacteriostatic effect on the cells at the concentration of 25 µg mL−1. The best antifungal activity has been detected in the presence of derivative XIII. Derivative XIII did also affect the morphology of hyphal tips of B. cinerea, which led to enhanced ramification of hyphae. Finally, the antimutagenic activity of derivatives was investigated. Significant antimutagenic activity was registered in case of derivative VIII. The number of induced revertants by mutagen [2-(5-nitrofuryl)acrylic acid] was decreased almost to the level of spontaneous revertants in the lowest applied concentration (50 µg per plate).

Prevalence of antibiotic-resistant coliform bacteria, Enterococcus spp. and Staphylococcus spp. in wastewater sewerage biofilm

OBJECTIVES Urban wastewater contains various micropollutants and a high number of different micro-organisms. Some bacteria in wastewater can attach to surfaces and form biofilm, which gives bacteria an advantage in the fight against environmental stresses. This work focused on analysis of bacterial communities in biofilms isolated from influent and effluent sewerage of a wastewater treatment plant (WWTP) in Bratislava, Slovakia. METHODS Detection of biofilm microbiota was performed by culture-independent and -dependent approaches. The composition of bacterial strains was detected by denaturing gradient gel electrophoresis fingerprinting coupled with construction of 16S rRNA clone libraries. Analysis of the concentration of antibiotics and the prevalence of antibiotic-resistant coliforms, Enterococcus spp. and Staphylococcus spp. in sewerage was also studied. RESULTS Biofilm collected at the inlet point was characterised primarily by the presence of Pseudomonas spp., Acinetobacter spp. and Janthinobacterium spp. clones, whilst members of the genus Pseudomonas were largely detected in biofilm isolated in outflow of the WWTP. Predominant antibiotics such as azithromycin, clarithromycin and ciprofloxacin were found in influent wastewater. The removal efficiency of these antibiotics, notably azithromycin and clarithromycin, was 30% in most cases. CONCLUSION The highest number of antibiotic-resistant bacteria, with a predominance of coliforms, was detected in samples of effluent biofilm. Multidrug-resistant strains in effluent biofilm showed very good biofilm-forming ability.