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Article

Evaluation of the Effectiveness of the Activated Sludge Process in the Elimination Both ATB-Resistant and ATB-Susceptible E. coli Strains

by
Iwona Beata Paśmionka
1,*,
Karol Bulski
1,
Piotr Herbut
2,3,
Elżbieta Boligłowa
1,
Frederico Márcio C. Vieira
3,
Gabriela Bonassa
4,
Marina Celant De Prá
4 and
Marcelo Bortoli
4
1
Department of Microbiology and Biomonitoring, Faculty of Agriculture and Economics, University of Ariculture in Krakow, 31-120 Kraków, Poland
2
Department of Rural Building, Faculty of Environmental Engineering and Land Surveying, University of Agriculture in Krakow, 31-120 Kraków, Poland
3
Grupo de Estudos em Biometeorologia—GEBIOMET (Biometeorology Study Group), Universidade Tecnológica Federal do Paraná (UTFPR), Estrada para Boa Esperança, km 04, Comunidade São Cristóvão, Dois Vizinhos 85660-000, Brazil
4
Grupo de Pesquisa em Gerenciamento e Tratamento de Resíduos—GETRAT, Universidade Tecnológica Federal do Paraná (UTFPR), Estrada para Boa Esperança, km 04, Comunidade São Cristóvão, Dois Vizinhos 85660-000, Brazil
*
Author to whom correspondence should be addressed.
Energies 2021, 14(18), 5868; https://0-doi-org.brum.beds.ac.uk/10.3390/en14185868
Submission received: 20 July 2021 / Revised: 13 September 2021 / Accepted: 14 September 2021 / Published: 16 September 2021
(This article belongs to the Special Issue Wastewater, Water and Energy)
Browse Figures
Versions Notes

Abstract

:
Water reuse is now becoming a global necessity. However, one of the drawbacks in releasing wastewater into the environment is some persistent pollutants that are not completely removed in wastewater treatment plant. Residual bacteria and antibiotics in the inflowing wastewater can contribute to the antibiotic resistance spread in the aquatic environment. This study determined the effectiveness of activated sludge process for fecal coliform bacteria elimination, and also the Escherichia coli resistance to antimicrobial agents as erythromycin, azithromycin, clarithromycin, ofloxacin, ciprofloxacin, trimethoprim, and metronidazole in treated wastewater. The research was carried out using the membrane filtration technique, and the susceptibility of isolates to antimicrobial agents was tested by the disc diffusion method. The concentrations of fecal coliform bacteria and Escherichia coli differed significantly depending on the seasonal period in which it was carried out. Despite up to 99% reduction in the number of sanitary indicators in biologically treated wastewater, 89% of E. coli isolates resistant to the tested antibiotics was found, while 100% of the isolates were susceptible to metronidazole. Most of the isolates showed resistance to trimethoprim, and the fewest isolates were resistant to ofloxacin, indicating that some strains may react differently to antibiotics.

1. Introduction

The constantly deepening world water deficit makes it necessary to ensure the appropriate quality of available water resources. The unquestionable benefits of wastewater treatment plants are followed by certain difficulties. The most important of which are the problems of wastewater sludge management and discharging to water bodies. Microbiological contamination is one of the basic threats to surface waters which are receivers of wastewater and potential sources of drinking water. Apart from physical and chemical pollutants, wastewater also contains numerous pathogenic and opportunistic microorganisms, mainly of intestinal origin. Raw wastewater is the largest reservoir of human intestinal bacteria. In addition, treated wastewater, even with the use of highly effective methods, may pose a serious microbiological threat to the receiving waters in terms of bacteriology. Therefore, one of the most important current technological challenges is effective wastewater treatment, which will ensure not only the appropriate physicochemical quality of the effluents, but also the microbiological condition that is safe in terms of sanitation.
Among the potential environmental threats related to the spread of waterborne microorganisms is the increasing phenomenon of antibiotic resistance. Wastewater is an important reservoir of antibiotic resistance genes [1,2], which also contain antibiotics and their metabolites, contributing to the selection pressure. This phenomenon leads to the elimination of susceptible bacterial strains and the selection of resistant ones that spread in the ecological niche that has emerged. It has been shown that in wastewater treatment processes based on the activated sludge method, a positive selection of bacteria resistant to antibiotics takes place [3,4,5,6], e.g., selection of E. coli isolates resistant to penicillins, fluoroquinolones, and trimethoprim-sulfamethoxazole [7,8]. Moreover, the transformation of plasmid DNA obtained from selected E. coli isolates from raw and purified wastewater indicates a possible transfer of resistance genes to β-lactam antibiotics and tetracycline [9]. More and more attention is devoted to understanding trends in acquired antibiotic resistance among bacteria living in the natural environment. There is a high probability that the traits of resistance can be transferred mainly as a result of horizontal gene transfer between bacterial populations from natural environments and the populations brought into the environment that are part of the saprophytic and pathogenic human microflora [10]. In this process, commensal bacteria are considered to be potential resistance gene transfer vectors.
Antimicrobial resistance poses a serious threat to the global public health system. Environmental contamination with pharmaceuticals increases as a result of improper or excessive use of antimicrobial substances and problems with their disposal from wastewater. As a consequence, more and more waters are contaminated with drugs, including antibiotics [11], leading to an increase in the number of antibiotic resistant bacterial strains.
In recent years, the role of wastewater treatment plants in transferring bacterial resistance to antibiotics has been widely discussed [12,13,14]. Antibiotics and antibiotic-resistant bacteria reach treatment plants with various municipal, hospital, and industrial wastewater. The high concentration of bacteria in the activated sludge promotes the processes of horizontal gene transfer. Due to the presence of pharmaceuticals, heavy metals, and other inhibitory compounds in the activated sludge reactor, there may be a selection pressure that favors resistance to antibiotics [15,16,17]. Despite the research intensification, the issue of the potential spread of antibiotic resistance through wastewater treatment plants remains unresolved.
Due to the possible impact of antibiotic substances on living organisms, measures should be taken to monitor the presence of these compounds in various environments. An important issue is also a thorough understanding of the ecology of bacteria resistant to antimicrobial agents. Microbiological indicators such as enteric sticks are commonly used in the sanitary assessment of the environment, and in particular, the number of Escherichia coli is monitored.
It is extremely important to analyze the presence of antibiotic resistance directly in the wastewater treatment plant, as well as in the treated wastewater, which is then discharged into surface waters. This allows the actual impact of the treatment plant on the spread of antibiotic resistance in the environment to be determined. The spread of antibiotic resistance is calling into question the future of many treatments. The status of antibiotic-resistant bacteria and genes that determine antibiotic resistance in environments receiving treated wastewater remains unclear. Therefore, the study focuses on the microbiological hazards of outflow from wastewater treatment plants, including the occurrence and selection of antibiotic-resistant isolates in wastewater treatment processes and the potential transfer of resistance features to the water environment of receivers.
The aim of the research was to determine the effectiveness of biological wastewater treatment in the elimination of fecal coliform bacteria (thermotolerant), including Escherichia coli, and to compare the susceptibility to selected antibiotics of Escherichia coli isolates present in treated wastewater at the WWTP in Oświęcim in Poland.

2. Materials and Methods

2.1. Treatment Plant Characteristics

The tests were conducted at the Municipal and Industrial Sewage Treatment Plant in Oświęcim (50°02′17.1″ N 19°19′13.8″ E). The treatment plant is located in south-eastern Lesser Poland and collects municipal wastewater from the town and commune of Oświęcim and industrial wastewater from the nearby Chemical Production Plants. The current amount of municipal wastewater is about 14,000 m3 per day and includes:
  • Domestic wastewater and wastewater from small production enterprises—11,320 m3 per day;
  • Infiltration waters—2300 m3 per day;
  • Wastewater delivered from septic tanks—380 m3 per day.
The amount of industrial wastewater from the Chemical Production Plants is approximately 26,400 m3 per day.
The composition of wastewater flowing into the treatment plant during the research period is shown in Table 1.
Initial treatment of municipal and industrial wastewater takes place in separate technological lines consisting of a grate, sand trap, and preliminary settling tanks. Post-production wastewater is additionally subjected to the process of neutralization and coagulation. After mixing with municipal wastewater in a 2:1 ratio, it is sent for biological treatment.
Biological treatment is based on the technology of low-loaded activated sludge. The biological system consists of an anaerobic reactor, four aeration reactors, three secondary radial settling tanks, a blower station, and an activated sludge pumping station (Figure 1).
The treated wastewater together with the activated sludge flows by gravity to three secondary radial settling tanks. In the secondary settling tank, the activated sludge is separated from the wastewater. The settled activated sludge is collected into the central funnel and flows to the reactor, from which it is returned to the process as recirculated sludge or is pumped out as excess sludge. The treated wastewater is discharged to the Macocha stream, which is a tributary of the Vistula River.

2.2. Sampling and Bacteriological Contamination Analysis

The research was conducted in the period from March 2020 to February 2021. The samples consisted of inflowing and biologically treated wastewater. Five hundred cubic centimeters of wastewater were collected in the spring, summer, autumn, and winter periods, in triplicate. A total of 24 wastewater samples were collected (12 inflowing wastewater samples and 12 treated wastewater samples), two samples per month. The inflowing wastewater samples representing industrial and raw municipal wastewater (mixed 2:1) were pretreated by sand removal process. The treated wastewater samples were collected at the end of the wastewater discharge channel to the receiver. Chilled samples were taken to the laboratory for immediate analysis.
The presence of the tested bacteria in the samples was assessed using the membrane filtration method on Endo agar (Emapol, Gdańsk, Poland) in accordance with the adopted standards [18]. The samples were filtered through 0.47-mm cellulose acetate membrane filters with a nominal pore size of 0.45 µm (Sartorius, Göttingen, Germany) using a Sartorius filtration kit and a Rocker 300 vacuum pump. Depending on the wastewater concentration, prior to filtration, 10-fold serial dilutions up to 1:1,000,000 in physiological NaCl solution were made. A maximum of 100 cm3 of the undiluted sample was filtered. Each volume was filtered in triplicate. The filters were transferred to 50-mm Petri dishes containing Endo agar and incubated at 44 °C. After 24 h of incubation, fecal coliforms were counted using the eCount Colony Counter (Heathrow Scientific) and the results were shown as CFU·100 cm−3 (CFU-colony forming units).

2.3. Escherichia coli Isolation and Identification

Pure cultures of E. coli were isolated from samples of treated wastewater in which the presence of thermotolerant coliform bacteria was detected. For this purpose, inoculation on Eosin-Methylene-Blue agar (Biocorp, Issoire, Poland) was used. After 24 h of incubation at 44 °C, purple-colored colonies with a green metallic luster (similar to Escherichia coli) were transferred to TBX agar (BTL, Warszawa, Poland). Tryptone Bile X-glucuronide Agar is a chromogenic medium on which E. coli grows at 44 °C in the form of blue-green colonies. Systematic position of isolates was determined based on biochemical properties [19] using API20E tests (Bio-Merieux, Marcy l’Étoile, France).

2.4. Antibiotic Resistance Testing

In order to investigate the susceptibility of E. coli isolates to antibiotics, disc diffusion tests (BIO-RAD, Hercules, CA, USA) were performed on Mueller–Hinton II medium (BTL, Warszawa, Poland). Sensitivity assessment and interpretation of results were performed according to the guidelines of KORLD (National Reference Center for Susceptibility Testing) and CLSI (Clinical and Laboratory Standards Institute) [20]. Inhibition diameters of growth were measured after 18–20 h of incubation at 37 °C. The results were compared with the breakpoint values provided by EUCAST (European Committee on Antimicrobial Susceptibility Testing) [21].
The discs used contained the antimicrobials most often detected in WWTPs: erythromycin (ERY 15 μg), azithromycin (AZM 15 μg), clarithromycin (CLR 15 μg), ofloxacin (OFX 5 μg), ciprofloxacin (CIP 5 μg), ampicillin (AMP 10 μg)), trimethoprim (TMP 5 μg), and metronidazole (MTR 50 μg) [22]. E. coli ATCC 25922 strain was used for quality control.

2.5. Data Analysis

In order to determine the significance of differences between the amount of fecal coliforms and E. coli isolated from wastewater, a one-way ANOVA was used, and Tukey’s test was used to verify the differences in the number of bacteria between seasons in each type of wastewater (differences significant for p < 0.05). One-way ANOVA and a Scheffe’s test (as the most conservative post hoc test, p < 0.05) were used to evaluate the differences between the percentages of resistance of E. coli isolates in the tested wastewater. The correlation between the number of tested microorganisms and the wastewater temperature was assessed using the Pearson correlation (p < 0.05). The tests were performed with Statistica v. 13.1 (StatSoft, Tulsa, OK, USA).

3. Results

Typically, the only monitoring tool used to assess the microbiological safety of reclaimed water is the total concentration of indicator organisms, including fecal coliforms (thermotolerant) and E. coli. Utilities and regulatory agencies rely on an assumed relationship between the indicator organism and pathogen survival/transport through wastewater treatment plants to ensure that the reclaimed water is safe for public use. While it is impossible to test reclaimed water for all possible pathogens, it is important that the indicator organisms used to ensure water quality correlate with a wide range of waterborne pathogens, such as bacteria, viruses, and protozoa.
Therefore, the number of fecal coliforms and Escherichia coli, including isolates resistant to antimicrobial agents, was determined as the basic indicator of bacterial contamination in sanitary analyses to assess the degree of contamination with fecal contamination.
In all analyzed wastewater samples, the presence of fecal coliforms, including E. coli, was found. Table 2 and Table 3 compare of the concentrations of fecal coliforms and E. coli in the inflowing and treated wastewater. The amount of antibiotic-resistant E. coli isolated in the treated wastewater is presented in Table 4.
During the research, the microbiological quality of the inflowing wastewaters was different. The amount of thermotolerant fecal coliforms ranged from 4.03 × 105 to 6.43 × 105 CFU·100 cm−3 (Table 2). E. coli ranged in the amount from 2.93 × 104 to 5.23 × 104 CFU·100 cm−3 (Table 3). The number of fecal coliform bacteria in the treated wastewater ranged from 3.03 × 103 to 13.53 × 103 CFU·100 cm−3, with the lowest concentrations observed in the autumn and winter, and the highest in the spring and summer (Table 2). The number of E. coli, including antibiotic-resistant isolates, was varied (Table 3 and Table 4). The summer and winter periods were characterized by the greatest variability in the concentrations of the analyzed indicators in the wastewater. This could be due to the share of runoff during sampling and the low temperature of the wastewater in winter.
Statistical analysis showed significant differences in the average number of fecal coliforms in the inflowing wastewater only during the summer and winter periods (p < 0.05). In the case of treated wastewater, there was a statistically significant difference in the average number of fecal coliforms between summer and other seasons of the year (p < 0.05). However, no significant differences were found between spring, autumn, and winter (p > 0.05) (Table 2).
With regard to E. coli, statistical analysis showed significant differences in the average number of these bacteria in incoming wastewater, between the summer season and other seasons of the year (p < 0.05). No significant differences were found between the spring, autumn and winter seasons (p > 0.05) (Table 3).
The question always arises whether the microorganism concentration in the inflowing wastewater affects the effectiveness of their removal in the biological treatment process, or whether it is primarily influenced by the design and operation of the treatment plant. In the conducted seasonal research series, the number of fecal coliforms and E. coli was reduced by over 99% (Table 2 and Table 3) and the levels of these indicators were within the requirements of the permit. However, in the treated wastewater, as many as 89% (610) of isolates resistant to seven out of eight selected antibiotics were found among E. coli (Table 4). Only 11% (76) of the isolates were susceptible to the action of all tested antibiotics, which may be of concern (Figure 2). The most effective was metronidazole, to which 100% of the isolates showed susceptibility. The greatest numbers of isolates were resistant to trimethoprim (68%) and ampicillin (56%), while the least to the effect of ofloxacin—21%. A high percentage of resistant isolates was also found for erythromycin (38%) and azithromycin (36%) (Table 5). Resistance variability also depends on the season, as observed. The greatest numbers of antibiotic-resistant E. coli were isolated in the summer and the least in the winter. In August, among 132 isolates, as many as 116 were resistant to at least one tested antibiotic. In February, among the 13 tested isolates, all showed resistance to trimethoprim and only 2 to erythromycin, azithromycin, clarithromycin, ofloxacin, ciprofloxacin, and ampicillin (Table 4). In the conducted studies, resistance to as many as 7 antibiotics was simultaneously demonstrated by 14% of isolates. However, the number of multi-antibiotic-resistant strains to 4 or more antibiotics was significantly lower than those resistant to 1, 2, or 3 antibiotics (Figure 2).
Taking into account the seasons, statistically significant differences were found between the percentages of E. coli resistance to antimicrobial agents. Significant seasonal differences were found for the antibiotics: erythromycin, azithromycin, clarithromycin, and ampicillin (p < 0.05). No significant differences between the percentages of resistance of E. coli isolates to antimicrobial agents in the seasons were observed to ofloxacin, ciprofloxacin, trimethoprim, and metronidazole (p > 0.05) (Table 4).

4. Discussion

The persistence of fecal coliforms and E. coli is related to the initial concentration of these microorganisms and the biological treatment effectiveness. Each subsequent stage of treatment can eliminate the concentration of indicators, depending on the characteristics of wastewater and key technological parameters. If the treatment system is highly effective, e.g., by using disinfection, the indicator concentrations may be below the detection limits [22,23,24,25].
Wastewater treatment plants operating on the basis of activated sludge technologies are characterized by different effectiveness in the elimination of sanitary indicators. In the conducted studies, the largest amount of coliform bacteria and Escherichia coli was observed in the inflowing sewage during the summer. However, in the process of biological treatment, their amount was reduced significantly above 90% in all research series. An equally high degree of reduction of fecal coliform bacteria was observed by Fars et al. [26].
Currently, more and more attention is paid to the participation of E. coli in the increase in antibiotic resistance in the aquatic environment. In the conducted studies, a total of 686 strains of E. coli were isolated from treated wastewater. The most common resistance was observed to trimethoprim (68%) and ampicillin (56%) (Table 5). Similar results were obtained by Piganto et al. [27] and Patoli et al. [28], considering ampicillin as the antibiotic to which E. coli bacteria isolated from water were the most resistant (88.89%; 22.71%; 18% resistant strains). In our research, a large percentage of resistant isolates was also found in the case of erythromycin and azithromycin. However, no resistance to metronidazole was observed (Table 5).
During the performed tests, the variability of resistance to antibiotics was slightly dependent on the season. The increase or decrease in resistance was according with the type of tested antibiotic, as observed (Table 4 and Table 5). This phenomenon can be related to various interacting factors. The tested wastewater had a specific chemical composition. This was a mixture of municipal and industrial wastewater (1:2) and contained various inhibitory substances of organic and inorganic origin that can affect bacteria. These are referred to as stressors [29,30,31]. Examples of stressors are antibiotic residues and their degradation products, which can shape the surviving microbial community in the wastewater treatment process. Because of this, different organisms or related groups have different tolerance degrees or defense mechanisms [32,33]. Thus, depending on the type of antibiotic that may be present in the wastewater treatment plant, different bacterial resistance behavior in the treated wastewater was observed. For example, literature data show that metronidazole is active against coliform bacteria [34]. Therefore, in the conducted studies it was included in the evaluation of E. coli resistance. As expected, none of the E. coli isolates showed resistance in the tests performed. However, the occurrence of metronidazole-resistant strains in subsequent analyzes cannot be predicted. The observed difference in resistance may also be related to the microbiota (mainly composed of human commensal bacteria) present during the study, which is mixed with bacteria of various origins. These microorganisms can colonize the activated sludge [35], in which the fraction of antibiotic-resistant bacteria can reach much more than 50%, at least within a given group (e.g., E. coli), e.g., as a result of horizontal gene transfer [35,36].
In the conducted studies, 11% of E. coli isolates were susceptible to all tested antibiotics. Unfortunately, the remaining isolates (89%) were resistant to one or more antibiotics. Resistance to as many as 7 antibiotics was demonstrated simultaneously by 14% of isolates (Figure 2). Sahm et al. [37] found that strains multi-antibiotic resistant to 4 or 5 antibiotics are much less than those that are resistant to one or two, which was confirmed by the conducted studies.
The fractions of antibiotic-resistant bacteria present in the wastewater and the antibiotic resistance genes in the aeration reactor came into contact with the activated sludge microorganisms. When subjected to the potential selection pressure of antibiotics, the potential for the spread of antibiotic resistance increased. Microbiota (including antibiotic-resistant bacteria) reaching the biological reactor is stimulated to compete with the activated sludge bacteria for the available organic matter. This intense metabolic activity creates an important dynamic for the bacterial community [38]. Changes in the bacterial community, the performance of antibiotic-resistant bacteria, and the success with which their genes spread to other bacteria are the key factors important to the transmission of antibiotic resistance [39]. Because wastewater treatment plants are directly connected to the aquatic environment and are not adapted to eliminate antibiotic resistance genes and antibiotic-resistant bacteria, they promote their spread among enteric and pathogenic bacteria [40,41,42].
The evaluation of antibiotic resistance of E. coli isolates indicates the presence of antibiotic-resistant bacteria in treated wastewater, even with the high degree of reduction in sanitary indicators. This results in a release of it to the aquatic environment and the spread of resistant strains. The obtained results contribute to the increased awareness of antibiotic-resistant bacteria spread through wastewater treatment plants, despite the satisfactory removal of other indicators (BOD5, COD, total nitrogen, total phosphorus, total suspended solids) [43]. In addition, they confirm the need for wastewater treatment techniques to prevent the antibiotic resistance spread, as recommended by the World Health Organization [44].

5. Conclusions

In the conducted studies, the concentrations of traditional bacterial indicators (fecal coliforms and E. coli) differed significantly according with the temporal wastewater samples. Despite almost 100% effectiveness of the biological treatment of wastewater in the elimination of fecal coliform bacteria, including E. coli, a fairly large percentage of E. coli isolates resistant to antimicrobial agents was found in the biologically treated wastewater. Perhaps in the process of wastewater disinfection, the number of strains carrying resistance genes into the water could be significantly reduced. From this study, it is evident that increased monitoring for alternative markers and pathogens is warranted in order to build a better database on the prevalence and reduction of intestinal bacteria, viruses, and parasites in reclaimed water. There should also be more discussion on the effectiveness of the process requirements compared to the reclaimed water quality targets.
The treated effluents monitored in WWTP in Oświęcim are not free from coliform bacteria, including antibiotic-resistant E. coli. This wastewater is discharged into the Vistula River, which means that public exposure to these waters carries some risk, although this level may be very low and quite acceptable for the majority of the population. Integrating microbiological monitoring with controls related to process design and operations can lead to a more robust approach to ensuring the safety of reclaimed water.

Author Contributions

Conceptualization, I.B.P. and K.B.; data curation, I.B.P. and K.B.; formal analysis, I.B.P., K.B., P.H. and E.B.; investigation, I.B.P.; methodology, I.B.P.; resources, I.B.P.; supervision, P.H. and E.B.; writing—original draft, I.B.P. and K.B.; writing—review and editing, I.B.P., K.B., P.H., E.B., F.M.C.V., G.B., M.C.D.P. and M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financed by Ministry of Science and Higher Education of the Republic of Poland and funded by Faculty of Environmental Engineering, University of Agriculture in Krakow, through project “Scientific subvention D014—Environmental Engineering, Mining and Energy”.

Conflicts of Interest

The authors declare no conflict of interest.

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Energies 14 05868 g001 550
Figure 1. Diagram of the biological wastewater treatment system in the Municipal and Industrial Wastewater Treatment Plant in Oświęcim, Poland.
Figure 1. Diagram of the biological wastewater treatment system in the Municipal and Industrial Wastewater Treatment Plant in Oświęcim, Poland.
Energies 14 05868 g001
Energies 14 05868 g002 550
Figure 2. Percentage of multi-antibiotic-resistant E. coli isolates.
Figure 2. Percentage of multi-antibiotic-resistant E. coli isolates.
Energies 14 05868 g002
Table
Table 1. Wastewater composition.
Table 1. Wastewater composition.
Sampling DatepHBOD5
(mg·dm−3)		COD
(mg·dm−3)		Total Suspended Solids
(mg·dm−3)		Total Nitrogen
(mg·dm−3)		Total Phosphorus
(mg·dm−3)
Spring7.7406.6466333746.98.44
Summer7.7454.7173325343.98.98
Autumn7.6232.1495617452.58.84
Winter7.8199.6688338850.16.18
Table
Table 2. The amount of fecal coliform bacteria in the inflowing and treated wastewater.
Table 2. The amount of fecal coliform bacteria in the inflowing and treated wastewater.
Sampling DateFaecal Coliform Bacteria (FC)The Degree of Reduction FC
(%)		Average Temperature (°C)
(CFU·100 cm−3)
Inflowing WastewaterTreated Wastewater
The Amount of FCAverage Number of FC with SD The Amount of FCAverage Number of FC with SDInflowing WastewaterTreated Wastewater
SpringMar3.7 × 1054.73 ± 1.1 × 105 ab *5.3 × 1037.13 ± 2.1 × 103 a98.513.7014.00
Apr4.6 × 1056.7 × 103
May5.9 × 1059.4 × 103
SummerJun6.1 × 1056.43 ± 3.1 × 105 b11.1 × 10313.53 ± 2.6 × 103 b97.918.3018.30
Jul6.5 × 10513.2 × 103
Aug6.7 × 10516.3 × 103
AutumnSep5.1 × 1054.70 ± 4.6 × 105 ab8.6 × 1035.57 ± 2.6 × 103 a98.814.7015.30
Oct4.8 × 1054.2 × 103
Nov4.2 × 1053.9 × 103
WinterDec3.8 × 1054.03 ± 5.9 × 105 a3.1 × 1033.03 ± 0.1 × 103 a99.39.7010.00
Jan4.7 × 1053.1 × 103
Feb3.6 × 1052.9 × 103
* Averages marked with the same letters are not significantly different by Tukey test (α = 0.05) (one-way ANOVA, separately for each type of wastewater between seasons).
Table
Table 3. The amount of Escherichia coli in the inflowing and treated wastewater.
Table 3. The amount of Escherichia coli in the inflowing and treated wastewater.
Sampling DateEscherichia coliThe Degree of Reduction E. coli
(%)		Average Temperature (°C)
(CFU·100 cm−3)
Inflowing WastewaterTreated Wastewater
The Amount of E. coliAverage Number of E. coli with SD The Amount of E. coliAverage Number of E. coli with SD Inflowing WastewaterTreated Wastewater
SpringMar3.0 × 1043.77 ± 0.9 × 104 a4459 ± 19.30 a99.8413.7014.00
Apr3.5 × 10453
May4.8 × 10481
SummerJun5.2 × 1045.23 ± 0.2 × 104 b94111 ± 19.43 b99.7918.3018.30
Jul5.1 × 104106
Aug5.4 × 104132
AutumnSep4.2 × 1043.63 ± 0.6 × 104 a7144 ± 24.27 a99.8814.7015.30
Oct3.6 × 10437
Nov3.1 × 10424
WinterDec2.9 × 1042.93 ± 0.2 × 104 a1515 ± 1.53 a99.959.7010.00
Jan3.1 × 10416
Feb2.8 × 10413
Averages marked with the same letters are not significantly different by Tukey test (α = 0.05) (one-way ANOVA, separately for each type of wastewater between seasons).
Table
Table 4. The amount of antibiotic-resistant E. coli isolated from treated wastewater.
Table 4. The amount of antibiotic-resistant E. coli isolated from treated wastewater.
Sampling DateTotal Amount of E. coli IsolatesNumber of E. coli Isolates Resistant to:
ERYAZMCLROFXCIPAMPTMPMTR
SpringMarch44171167823270
April531914681028330
May8131209121644500
SummerJune94454834282764580
July106515438313171680
August1326468483939891160
AutumnSeptember7115131081433420
October378864718220
November245443411150
WinterDecember15222022100
January16333533140
February13222222130
Table
Table 5. Amount of antibiotic susceptible and resistant E. coli in the treated wastewater.
Table 5. Amount of antibiotic susceptible and resistant E. coli in the treated wastewater.
AntibioticNumber of Susceptible StrainsNumber of Resistant StrainsResistance (%)
ERY42426238
AZM43924736
CLR51816824
OFX53914721
CIP52316323
AMP29838856
TMP21846868
MTR6860-
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Paśmionka, I.B.; Bulski, K.; Herbut, P.; Boligłowa, E.; Vieira, F.M.C.; Bonassa, G.; Prá, M.C.D.; Bortoli, M. Evaluation of the Effectiveness of the Activated Sludge Process in the Elimination Both ATB-Resistant and ATB-Susceptible E. coli Strains. Energies 2021, 14, 5868. https://0-doi-org.brum.beds.ac.uk/10.3390/en14185868

AMA Style

Paśmionka IB, Bulski K, Herbut P, Boligłowa E, Vieira FMC, Bonassa G, Prá MCD, Bortoli M. Evaluation of the Effectiveness of the Activated Sludge Process in the Elimination Both ATB-Resistant and ATB-Susceptible E. coli Strains. Energies. 2021; 14(18):5868. https://0-doi-org.brum.beds.ac.uk/10.3390/en14185868

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Paśmionka, Iwona Beata, Karol Bulski, Piotr Herbut, Elżbieta Boligłowa, Frederico Márcio C. Vieira, Gabriela Bonassa, Marina Celant De Prá, and Marcelo Bortoli. 2021. "Evaluation of the Effectiveness of the Activated Sludge Process in the Elimination Both ATB-Resistant and ATB-Susceptible E. coli Strains" Energies 14, no. 18: 5868. https://0-doi-org.brum.beds.ac.uk/10.3390/en14185868

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