Effect of Wastewater Treatment on Bacterial Community, Antibiotic-Resistant Bacteria and Endoparasites

Wastewater and wastewater treatment plants serve as urban reservoirs of pathogenic microorganisms. Wastewaters frequently contain bacteria, antibiotic-resistant bacteria, and developmental stages of parasites with significant zoonotic potential. Five wastewater treatment plants in the central part of Slovakia were investigated to determine the effect of treatment on bacterial community, antibiotic-resistant bacteria, and the occurrence of helminth eggs. Although all monitored chemical factors (chemical oxygen demand, biochemical oxygen demand, N-NH4, total nitrogen, and total phosphorus) in the effluent were in line with the legislative standards for discharge into public waterways, the results of minimal inhibitory concentrations show that reclaimed water harbors E. coli resistant to several commonly used antibiotics (ampicillin, piperacillin, and tazobactam, combine ampicillin and sulbactam, cefotaxime, tetracycline). The presence of endoparasite developmental stages in wastewater and sludge (Ascaris spp., Hymenolepis nana, eggs from the Ancylostomatidae family, Giardia duodenalis) indicates potential health risks for humans and workers at these sites. Treatment such as composting before applying sludge to land is necessary to reduce human pathogens.


Introduction
Wastewater and wastewater treatment plants serve as urban reservoirs for pathogenic microorganisms [1,2]. Wastewaters frequently contain developmental stages of parasites with likely zoonotic potential. Many viruses and bacteria are devitalized during the treatment process in a wastewater treatment plant, but sometimes not entirely [3,4].
Frequent use of antibiotics in human and veterinary medicine leads to the spread of antibiotic-resistant bacteria into the environment. Antibiotic-resistant strains enter the environment through human feces and the liquid manure of animals. Antibiotics consumption and the increase of microorganisms' resistance can be seen by higher concentrations in wastewater, agriculture, livestock farming, and the human population [5][6][7]. Antibioticresistant bacteria and their genes are significantly emerging environmental pollutants. lipids, and in the nematode eggs also chitin [39]. All these pathogens are potentially dangerous for public health because the infection dose is low, and in some parasites, it can be accounted for by only one egg [40][41][42]. Wang et al. [20] reported that conventional onsite wastewater treatment systems could potentially contribute to the transmission of infectious diseases caused by waterborne pathogenic microorganisms and become an essential human health concern. On the other hand, sewage sludge can be a valuable source of organic matter for agriculture as it contains functional agrochemical-nutrient components applicable to the soil. Sludge has a high amount of organically bound nitrogen and phosphorus [43,44]. However, it also contains components that may pose a risk to the soil, water, and the human food chain, particularly at higher concentrations. Thus, this material requires qualified processing and utilization to prevent soil contamination and avoid the accumulation of risk components in the soil.
In the Slovak Republic, the annual production of sludge reaches close to 340 thousand tons. This material is included within the category of special wastes [45], which must be safely disposed of mainly due to the presence of pathogens. On the other hand, sludge is a valuable source of nitrogen, phosphorus, potassium, and some trace elements.
This work aimed to study the effect of wastewater treatment on bacterial communities, antibiotic-resistant bacteria, and the occurrence of developmental stages of endoparasites.

Description of Investigated Wastewater Treatment Plants
Five wastewater treatment plants (WWTPs) located in central Slovakia were investigated to determine the effect of treatment on bacterial communities, antibiotic-resistant bacteria, and helminth eggs. This study was performed in five wastewater treatment plants that remain anonymous and are labeled as "WWTP A-E". All wastewater treatment plants receive municipal wastewater and discharge the effluents into streams or rivers. The characteristics of studied wastewater treatment plants are described in Table 1. The generated sludge was treated by aerobic stabilization (WWTP B) or anaerobic stabilization (WWTP C) and applied to the compost. Sludge from WWTP A, D, and E was not treated in the plants. Unstabilized raw sludge in WWTP B was treated by aerobic stabilization. WWTP B was shut down during the monitored period, so obtaining samples from the influent portion was not possible.

Sample Collection
Samples were collected in the summer of 2020 (June-August). During the tested period, 9 samplings were performed and 3 samples were taken from each sampling place (influent, effluent, sludge).
Two liters of influent and effluent samples were collected to chemically clean bottles for the physicochemical evaluation, and 1 L was placed into a sterile bottle for microbiological and parasitological examination. A total of 2 L of raw sludge and 1 kg of treated sludge were collected. Samples were stored without any conservation at 4 • C and transferred to the laboratory for microbiological, parasitological, and chemical examination, performed within 24-48 h.

Chemical Examination of Samples
Chemical examination of wastewater (input and output) and sewage sludge included determination of pH, the levels of total nitrogen (N t ), water-soluble ammonium (NH 4 -N), total phosphorus (P t ), and chemical oxygen demand (COD Mn ). According to STN ISO 10523 [46], the pH was determined with a pH-meter (HACH Company, Loveland, CO, USA) and a WATERPROOF pH Tester 30. COD Mn was determined by oxidation with KMnO 4 according to STN EN ISO 8467 [47] and NH 4 -N by titration [48,49]. A portion of samples for N t determinations was digested using a HACH-Digesdahl apparatus (HACH Company, Loveland, CO, USA). N t was distilled with NaOH (40%) [50,51], and P t was determined by the vanadomolybdate method [52,53]. Efficiency of chemical pollutants was calculated according to the formula: According to National Regulation SR [54], the biological process of wastewater treatment is focused only on the reduction of chemical pollutants, which have to meet the maximal limit of chemical factors in the effluent grab samples of wastewater discharged as described in Table 2. Table 2. Maximal limit of chemical factors in the effluent grab samples of wastewater discharged to the recipient rivers [54]. N t -total nitrogen; NH 4 -N-water-soluble ammonium nitrogen; P t -total phosphorus; COD Mn -chemical oxygen demand; IS-insoluble substances; − means no maximal limit.

Isolation of Bacteria
The influent, effluent, and sludge wastewater samples were prepared by a serial dilution from 10 1 to 10 6 . All samples were examined in duplicate. Determination of relevant bacterial counts (total count of bacteria, coliform bacteria, fecal coliform bacteria, and fecal enterococci) was carried out in compliance with the Slovak Republic Government Regulation [55]. The total bacterial count (TCB) was determined according to STN EN ISO 6222 [56]. The pour-plate method on meat-peptone agar was followed by aerobic cultivation at 37 • C for 24 h for bacteria determination.
Coliform bacteria (CB) and fecal coliform bacteria (FCB) were cultivated according to STN EN ISO 9308-1 [57] using Endo agar (HiMedia, Mumbai, India) and incubated for 24 h at 37 and 43 • C, respectively. Then, the characteristic colonies were counted. A lactose fermentation test confirmed coliform bacteria presence.
Determination of fecal enterococci counts (FE) was carried out according to STN EN ISO 7899-2 [58]. The cultivation of respective bacteria was performed on Slanetz Bartley agar (Merck, Darmstadt, Germany) at 37 • C for 48 h.

Parasitological Examination of Samples
To determine protozoa (oo) cysts and helminth eggs count in influent and effluent samples from WWTP and sewage sludge, 50 mL from each 1 L sample was taken and examined by sedimentation and flotation techniques [62].
The dry sludge samples were surveyed according to Kazacos [63]. Briefly, 100 g of the pooled sludge sample, 100 mL of water, and 0.5 mL of Tween 40 were mixed and decanted for 10 min. Subsequently, the samples were sieved and replenished with 1000 mL of water. After 1 h of sedimentation, the soil samples were centrifuged (Eppendorf 5804, Hamburg, Germany) and then floated with sucrose flotation solution (specific density of 1.3). Samples were examined under the light microscope at 20× and 40× magnification (Leica Microsystems, DM 5000B light microscope, Wetzlar, Germany) to detect the presence of protozoa (oo) cysts and helminth eggs.

Results
WWTPs (A, D, E) are small, representing up to 10,000 residents. Here, the regularly monitored value is only one chemical indicator, i.e., CODMn (70 mg/L in grab samples or 135 mg/L in composite samples). In WWTP D and WWTP E, these values exceeded the norm (180 mg/L) and in WWTP E, this value exceeded the norm more than twice. Furthermore, in the biggest WWTP C, the CODMn was up to 282.8 mg/L, where only 125 mg/L is officially allowed. WWTP B was in reconstruction, and wastewater flowed directly to the river, and the COD level was exceeded twice Here the maximal limit was 140 mg/L (Table 3). WWTP-wastewater treatment plant; N t -total nitrogen; NH 4 -N-water-soluble ammonium nitrogen; P t -total phosphorus; COD Mn -chemical oxygen demand; IS-insoluble substances.
After mechanical and biological treatment in WWTPs, the effluent's chemical factors decreased within the limit in every monitored wastewater treatment plant.
The results of the microbial evaluation and the percentage of efficiency in the individual examined wastewater treatment plants are described in Figure 1. The log10 in the total number of microorganisms in individual wastewater treatment plants ranged from 7 to 5.4 in influent and from 6 to 3.4 in the effluent, representing a decrease from 99 to 89%. The total coliform bacteria concentration in the sludge varied from 7.6 to 5.7. The log10 of coliform bacteria ranged between 6.9 to 4 in the influent and in the effluent from 6.4 to 3.4, representing a 68 to 99% purification efficiency. The fecal coliform bacteria were 4.5 to 6 at the influent and from 2.6 to 4.9 in the effluent to express 95 to 99% efficiency. The log10 of fecal enterococci ranged from 4 to 4.4 in the influent and 2.2 to 4.2 in the effluent, thus showing the efficiency from 79 to 99%. The concentration of the monitored bacteria in the sludge was approximately the same as in the samples from the wastewater influent.
A modified microdilution method with the VetMIC panel detected antimicrobial resistance in 142 E. coli strains. The highest incidence of beta-lactamase resistance was observed for ampicillin (60% and MIC GX 18.9 mg/L in sludge samples), which was followed by ampicillin and sulbactam (35.21% and MIC GX 7.8 mg/L), tetracycline (20% in influent and 40% in effluent, MIC GX 3.8 mg/L), piperacillin and tazobactam (8.6%, MIC GX 2.2 mg/L in influent). MICs of cefuroxime and cefotaxime were 5.8 in influent and 5.6 mg/L (in effluent); tigecycline, trimethoprim and sulfonamides was about 5.6% (in effluent). The percentage of resistance to ceftazidime, tobramycin, amikacin, ciprofloxacin was 2.87% in influent and effluent components, and sludge showed low resistance to those antibiotics. Resistance to meropenem, sulbactam, cefepime, meropenem, gentamicin, and nitrofurantoin was 0% in all wastewater and sludge samples (Figure 2). 7 to 5.4 in influent and from 6 to 3.4 in the effluent, representing a decrease from 99 to 89%. The total coliform bacteria concentration in the sludge varied from 7.6 to 5.7. The log10 of coliform bacteria ranged between 6.9 to 4 in the influent and in the effluent from 6.4 to 3.4, representing a 68 to 99% purification efficiency. The fecal coliform bacteria were 4.5 to 6 at the influent and from 2.6 to 4.9 in the effluent to express 95 to 99% efficiency. The log10 of fecal enterococci ranged from 4 to 4.4 in the influent and 2.2 to 4.2 in the effluent, thus showing the efficiency from 79 to 99%. The concentration of the monitored bacteria in the sludge was approximately the same as in the samples from the wastewater influent. A modified microdilution method with the VetMIC panel detected antimicrobial resistance in 142 E. coli strains. The highest incidence of beta-lactamase resistance was observed for ampicillin (60% and MIC GX 18.9 mg/L in sludge samples), which was followed by ampicillin and sulbactam (35.21% and MIC GX 7.8 mg/L), tetracycline (20% in influent and 40% in effluent, MIC GX 3.8 mg/L), piperacillin and tazobactam (8.6%, MIC GX 2.2 mg/L in influent). MICs of cefuroxime and cefotaxime were 5.8 in influent and 5.6 mg/L (in effluent); tigecycline, trimethoprim and sulfonamides was about 5.6% (in effluent). The percentage of resistance to ceftazidime, tobramycin, amikacin, ciprofloxacin was 2.87% in influent and effluent components, and sludge showed low resistance to those antibiotics. Resistance to meropenem, sulbactam, cefepime, meropenem, gentamicin, and nitrofurantoin was 0% in all wastewater and sludge samples (Figure 2). Among all 142 investigated E. coli strains, the high penicillinase phenotype was confirmed in 9 strains (12.68%) and 11.27% of carbapenemases (detected in two strains from the influent portion). Three phenotypes were penicillinase low (4.23%). An AGL AAC(6′) phenotype was also detected in samples. Multi-drug resistance was confirmed in one sample (Figure 3). Based on the MIC phenotypic identification of E. coli strains, 12.5% were high-level resistance penicillinase occurrence, 4% low-level, 1.4% were multiresistant isolates, and 1.4% were aminoglycosides AAC(6′)I. The ESBLs were not detected in isolated Among all 142 investigated E. coli strains, the high penicillinase phenotype was confirmed in 9 strains (12.68%) and 11.27% of carbapenemases (detected in two strains from the influent portion). Three phenotypes were penicillinase low (4.23%). An AGL AAC(6 ) phenotype was also detected in samples. Multi-drug resistance was confirmed in one sample (Figure 3). Based on the MIC phenotypic identification of E. coli strains, 12.5% were high-level resistance penicillinase occurrence, 4% low-level, 1.4% were multiresistant isolates, and 1.4% were aminoglycosides AAC(6 )I. The ESBLs were not detected in isolated E. coli strains. Among all 142 investigated E. coli strains, the high penicillinase phenotype was confirmed in 9 strains (12.68%) and 11.27% of carbapenemases (detected in two strains from the influent portion). Three phenotypes were penicillinase low (4.23%). An AGL AAC(6′) phenotype was also detected in samples. Multi-drug resistance was confirmed in one sample (Figure 3). Based on the MIC phenotypic identification of E. coli strains, 12.5% were high-level resistance penicillinase occurrence, 4% low-level, 1.4% were multiresistant isolates, and 1.4% were aminoglycosides AAC(6′)I. The ESBLs were not detected in isolated E. coli strains. The results obtained from the parasitological examination are presented in Table 4. Giardia duodenalis was detected only in WWTP C influent section collected in June 2020. No other cysts were observed by microscopy in water or the sludge.  The results obtained from the parasitological examination are presented in Table 4. Giardia duodenalis was detected only in WWTP C influent section collected in June 2020. No other cysts were observed by microscopy in water or the sludge. There were no helminth eggs found in effluent portions. Ascaris spp. eggs were confirmed in influent from WWTP A, B, and E. No Ascaris spp. eggs were detected in influent from WWTP C and D. It was impossible to differentiate between the species since only the optical microscopy detection was carried out. Hymenolepis nana eggs and eggs from the family Ancylostomatidae were also found in the influent portion. The most prevalent helminth eggs in sludge were Ascaris spp. eggs and eggs from the family Ancylostomatidae (Table 4).

Discussion
The treatment process in the WWTPs is primarily focused on the removal of chemical pollutants (COD, BOD5, N-NH4, total nitrogen, and total phosphorus) [54], which are checked in effluent values as a preventive measure against river pollution (eutrophication of water) negatively impacting river ecosystems [42,64].
During the studied period, almost all chemical factors in effluent from monitored WWTPs were in line with the standards and regulations for discharge into public waterways.
COD, the measure of the oxygen equivalent of the organic matter and microorganisms in the wastewater, was higher in smaller and bigger WWTPs and exceeded the maximum level of 125 mg/L. In untreated domestic wastewater, COD usually ranges between 250 and 1000 mg/L [65].
By comparing the results of chemical parameters and microbiological analyses in the effluent, the higher efficiency of wastewater treatment in WWTP A, C, and D is visible. Almost all monitored microorganisms here decreased by 2 to 3 logarithmic orders. The concentration of microorganisms in the sludge in individual WWTPs was comparable or slightly higher than at the influent. A comparison of the number of microorganisms in the sludge of the monitored WWTPs found no significant differences (ranging from 6-7 log in TCB, CB, and 4.5-6 log in FCB and FE).
The number of coliform bacteria in effluent ranged from 3.4 to 6.3 log CFU·mL −1 which correlates with results of other works investigating wastewater in Slovak and Czech Republic (3.02-4.94 log CFU·mL −1 ) [66]. In effluent wastewater from hospitals, the concentrations of a number of coliform bacteria can reach 7.18 log CFU·mL −1 [67], which is in two orders higher when compared to the domestic and municipal WWTP, highlighting the importance of separated wastewater and sludge collection and treatment.
Interestingly, the effectiveness of the treatment process in small WWTP (up to 2000 recipient) is less than in big WWTP (up to 100,000 recipient). For example, in the WWTP E, the wastewater from 900 recipients had the highest concentration in chemical parameters and microbial concentration in influent decreased only by 0.5 log order. This signals low effectiveness of cleanability and possible failure in the treatment process. In addition, larger WTTPs employ more professional staff, the cleaning process is regularly controlled and inspected (including self-inspections), and the monitored parameters in the effluent are set up to be more strict.
Although the success of treatment by comparing the concentration of chemical parameters, microorganisms and parasites in effluent has been relatively high, it still poses a risk to rivers' pollution and habitats. It is not proven that the treatment is sufficient to remove antibiotic-resistant bacteria. It is known that some of these bacteria can carry resistance genes either in their core genome or on mobile genetic elements. These genes can be transferred from one bacterium to another via horizontal gene transfer under selection pressure [68]. There are also possibilities for plasmid transfer in wastewater treatment plants and surface waters [69,70].
In a recent study, the following E. coli resistances were detected: more than 50% to ampicillin, 35% were resistant to ampicillin and sulbactam, 20% to tetracycline, and 8.6% to piperacillin and tazobactam. E. coli isolates from the effluent also showed resistance to cefotaxime, cefepime, cefuroxime, tigecycline, trimethoprim, and sulfonamides (around 5%). There were only minor differences between antibiotic resistance results from influent, effluent, and sludge.
We confirmed E. coli strains the Penicillinase: high, carbapenemase, Penicillinase: low, and AGL AAC(6 ) phenotypes. A multidrug-resistant E. coli phenotype was detected in one sample.
In a previous study, Gregová and Kmet' [5] detected that the production of extendedspectrum β-lactamases in coliform isolates was encoded mainly by blaTEM, blaCTX-M-2, and blaCTX-M-8/25 genes. About 62% of resistant strains contained a combination of two or more extended-spectrum beta-lactamases (ESBL) genes.
Lépesová et al. [67] have found that more than half of the coliform bacteria from hospital wastewater were multidrug resistant and possessed a strong biofilm-formation ability.
A high concentration of the monitored bacteria and antibiotic-resistant E. coli isolates was found in the sludge, which still poses a risk of spreading microorganisms through the direct incorporation of sludge into agricultural land. However, the above indicates that sludge produced during municipal wastewater treatment must be subjected to additional processing to improve its hygiene level. Therefore, composting constitutes an economically advantageous and hygienically effective method of sludge processing. In addition, processing and organic recycling wastes by composting must ensure a product that fulfills a range of water management and hygiene requirements and complies with the principles of waste legislation [45].
Still, wastewater treatment plants play a vital role in minimizing the discharge of many water pollutants, including antibiotics [75,76]. Degradation of hormones, pesticides, antibiotics, antihistamines, and drugs is limited, and as a result, they commonly end up in the aquatic environment. Runoff and wastewater discharges may also contribute to the spread of organic and inorganic nutrients that may boost the growth and proliferation of indigenous or introduced pathogens.
Ascaris spp. eggs, viable eggs from the Ancylostomatidae family, and viable H. nana eggs were found in influent. This indicates that human feces and domestic animal excreta might contaminate wastewater treatment systems. During mechanical and biological treatment processes in the studied WWTPs, mainly bacteria, but not the endoparasites' developmental stages, were devitalized. Therefore, all above-mentioned helminths egg sediment and concentrate were in the sludge with suspended particles. In the case of poor sludge treatment, they again re-enter the environment and pose potential hygiene, epidemiological or epizootiological risks [19,36,77]. Finally, we can state that the helminth eggs in the sludge indicate the overall health status and the incidence of diseases in the population monitored in the WWTP area.
An exciting finding was confirmation of G. duodenalis cysts influent. No cysts were found in the effluent stage and sludge. However, information about the presence of protozoan pathogens is limited due to the inconsistencies in sampling as well as concentration and recovery procedures [44]. Despite our findings, it is necessary to pay special attention to this parasite because it has great zoonotic potential. G. duodenalis (syn. G. intestinalis, G. lamblia) is a unicellular parasite causing gastrointestinal disorders in a wide range of hosts, including wild and companion animals as well as humans. It is one of the most spread parasitic diseases in man, who can become infected via the fecal-oral route through contaminated water ("water-borne") and food ("food-borne"), or by direct transmission from host to host. This parasitic disease is classified as neglected and occurs more frequently in areas with poor hygiene standards, where children are the most affected population.
Similarly, Dudlová et al. [78] in east Slovakia studied the incidence of endoparasite germ's developmental stages (cysts, oocysts, protozoa, and helminth eggs) as an indirect monitoring factor for endoparasite circulation in raw municipal wastewater, sludge, and biologically cleaned wastewater. The raw wastewater contained cysts of Giardia spp., and Entamoeba spp., the helminth eggs Ascaris spp., and strongyle-type eggs. No protozoa cysts or helminth eggs were found in the treated wastewater. The highest occurrence of endoparasite developmental stages was detected in drained stabilized sludge. Protozoan (oo) cysts (Giardia spp., Cryptosporidium spp., Entamoeba spp.) and helminth eggs (Ascaris spp., Trichuris spp., Taenia spp., Hymenolepis spp., or strongyle-type eggs) were found. In drained and stabilized sludge, the eggs of Capillaria spp. and Toxocara spp. were also detected. In comparison with our results, differences in parasite composition can be because wastewater treatment plants in eastern Slovakia are located close to territories with low hygiene standards and a significant presence of residents belonging to marginalized populations. On the other hand, Amoah et al. [79], Chaoua et al. [80], and others have reported that wastewater treatment plants are not fully capable of helminth eggs removal from the water.
We confirmed that sedimentation is an effective removal mechanism for the helminth eggs. Similar results are reported by Kansiime et al. [81] and Dai et al. [82]. In the lowquality water, the eggs are incorporated into particle flocs with different settling velocities, and the settling rate of eggs and particles is closely associated [83]. Environmental conditions can also affect the survival of the pathogen in wastewater. For example, the rate of removing bacterial indicators (e.g., Salmonella and fecal coliform bacteria) is higher in summer than in winter [84,85]. UV radiation by sunlight is an effective mechanism for pathogen removal, mainly in open-water treatment wetlands. Temperature, pH, insolation, and solar radiation influence the survival of A. suum eggs [86][87][88].
Based on our results, we can conclude that the use of untreated or insufficiently treated wastewater poses many risks. The use of untreated wastewater is also associated with a higher infection intensity, especially for helminth infection. Ensink et al. [89] found a significantly increased risk of A. lumbricoides, hookworm, and T. trichiura infection in farming communities irrigating with wastewater. Aquatic recipients contaminated with wastewater containing helminth eggs also have a high concentration of suspended particles resulting in flocculation of the suspended material, including eggs [83].

Conclusions
Wastewater treatment plants play a vital role in minimizing the discharge of many water pollutants and the protection of the environment. The results show that the pattern of resistance in E. coli isolates found in monitored WWTPs is comparable with resistance seen at the clinical health care level. Ampicillin (beta-lactams), gentamicin (aminoglycoside), and ciprofloxacin (fluoroquinolones) are frequently prescribed drugs in Slovakia.
However, the main wastewater and sludge reuse limitation is contamination with helminth eggs due to its low infective dose and prolonged survival in the environment. In this study, several parasite species were identified in influent segments and sludge, where nematodes of human and animal origin were predominantly identified. In addition, the wastewater and sludge indicates potential health risks for humans and workers at these sites.