Antibiotic resistance in wastewater, does the context matter? Poland and Portugal as a case study

Abstract Antibiotic resistance has been considered a major human health threat that may endanger the success of medicine. Recent studies have unveiled worldwide asymmetries of antibiotic resistance occurrence, being factors as diverse as climate, socioeconomic, or antibiotic use possible drivers of such asymmetric distribution. In Europe, where clinical antibiotic resistance is surveyed for more than 20 years, the European Center for Disease Prevention and Control (ECDC) consistently describes an increasing gradient from North-to-South and from West-to-East. This observation motivated the current perspective paper aiming to qualitatively compare two countries located at the extreme latitude of Europe and also at distant longitude – Poland in the Central-East region and Portugal in the South-West. Both countries have been among those with the highest prevalence of antibiotic resistance in clinical settings, although as it is discussed, climate, socioeconomic factors, and antibiotic use are different. In general, in Poland higher antibiotic consumption and resistance prevalence is observed, mainly at the community level, when compared to Portugal. However, in Portugal, treated wastewater may hold identical or slightly higher resistance loads. Based on these observations, it is discussed how different factors may influence the abundance of antibiotics, antibiotic resistant bacteria, and genes in wastewater before and after treatment.


Introduction
Antibiotic resistance has become a serious threat to human health, mainly through opportunistic infections in patients under health care due to any other pathology (Fair & Tor, 2014). However, antibiotic resistance is not a strict health care issue, as it is spread among healthy people, domestic and farm animals, wildlife, and the natural environments, in particular water and soil (Hernando-Amado et al., 2019;McEwen & Collignon, 2018). The implications of the wide dissemination and unavoidable contact with antibiotic resistant bacteria (ARB) depend largely on the complex context where it occurs, from the socioeconomic and climate conditions to other factors still poorly understood (e.g., the effects of pollution or the ecology and biodiversity of distinct world regions). This is nicely discussed and evidenced in Collignon et al. (2018), who investigated the potential association of anthropological and socioeconomic factors. The study involved data of prevalence of third-generation cephalosporin (e.g., ceftazidime, cefotaxime), fluoroquinolone, and carbapenem resistance in Escherichia coli and in Klebsiella spp., for which was also used the prevalence of methicillin-resistance in Staphylococcus aureus. The data was collected from scientific literature, the database ResistanceMap (https://resistancemap.cddep.org/), and the antimicrobial resistance report published by the World Health Organization (WHO) in 2014 from 73 to 103 countries (depending on data availability) distributed over the five continents. Important variables that Collignon et al. (2018) concluded to be related to resistance included temperature and socioeconomic factors such as governance, gross domestic product, infrastructures, education, and health-care expenditure. Indeed, the database ResistanceMap, which combines information from distinct data sources (EARS-Net, GLASS, CHINET, among others) offers a worldwide overview of the contrasts on the clinical prevalence of antibiotic resistance in some important human pathogens. In general, Russia and Asian countries, a few African countries for which data is available, and some Latin American countries are those with the highest prevalence values (% of resistant invasive isolates), followed by Europe, North America, and Oceania (https://resistancemap.cddep.org/ accessed January 7, 2021). Despite the important gaps that may be noticed for some world regions, existing clinical surveys offer an outlook of the geographic trends of antibiotic resistance prevalence. This situation contrasts with the current knowledge of antibiotic resistance occurrence in the environment, typically unsystematic and scarce. The most studied environmental compartment is wastewater, and the metagenomics analysis of raw wastewater has been proposed as a method of community surveillance (Aarestrup & Woolhouse, 2020). This approach was used to analyze raw wastewater collected in 79 sites in 60 countries. The pattern of antibiotic resistance was similar to that described above for clinical isolates, with a clear separation between Europe, North America, and Oceania, with lower antibiotic resistance gene relative abundance than Africa, Asia, and South America (Hendriksen et al. 2019). Although Europe is in the low-prevalence group, it is quite asymmetric in terms of resistance prevalence. Consistently, in clinical settings, a general increase of antibiotic resistance prevalence is observed from North-to-South and from West-to-East, particularly for the Gram-negative bacteria (ECDC, 2020). This same pattern was observed in raw and treated wastewater from seven countries across this gradient (P€ arn€ anen et al. 2019). In this study, the resistance determinants measured by polymerase chain reaction (PCR) presented higher relative abundance in samples from Southern countries, in both raw wastewater and final effluents. These values were not significantly correlated with the concentration of antibiotic residues detected in the same samples, although, in general, these presented higher concentrations in the Southern than in Northern countries (Rodriguez-Mozaz et al., 2020). The authors concluded that, besides the antibiotic use, the temperature which is higher in Southern countries might also explain the described gradients of antibiotic resistance (P€ arn€ anen et al. 2019). Indeed, different publications have been showing that higher temperature may be a driver for antibiotic resistance increase (MacFadden et al., 2018;McGough et al., 2020;P€ arn€ anen et al. 2019).
The previous considerations and findings were major motivations to proceed with a literature and database-supported study comparing two European countries, Poland (Central-East Europe) and Portugal (South-West Europe), with contrasting climate conditions and placed among those with the highest resistance prevalence in clinical settings (ECDC, 2020). In a study analyzing the attributable deaths and disability-adjusted life-years (DALYs) caused by infections with ARB in European Union (EU) and European Economic Area (EEA), modeling analysis positioned both Poland (position 8) and Portugal (position 4) in the group of nine countries with a worse situation than the EU/EEA average, with about two times higher burden of ARB infections in Portugal than in Poland (24 021 vs. 41 069 reported cases, 1 158 vs. 2 218 attributable deaths), and also higher DALYs per 100 000 population ($240 vs. $190) (Cassini et al. 2019). This preliminary overview drove the definition of the objectives of the current analysis: 1) compare both countries regarding socioeconomic and climate conditions, antibiotic use in animal farming and human medicine, and prevalence of resistance in clinical pathogens; 2) compare the occurrence of ARB, antibiotic resistance genes (ARGs) and antibiotic residues in both countries, and infer about the fitness of these biological contaminants in wastewater.

Comparison of socioeconomic, geographic, and climate conditions
Poland and Portugal are located at latitude extremes of Europe, Central-East and Southern-West, respectively, having Poland an area approximately three times larger than Portugal (Table 1). The same is observed in terms of total population ($38.4 vs. $10.3 million, respectively in Poland and Portugal), making the average population density of the two countries similar (123 vs. 111 inhabitants/km 2 ) ( Table 1).
The different geographic locations influence the climate and weather conditions, as average temperature, global solar radiation, and precipitation (Table 1). According to the K€ oppen-Geiger climate classification (http://koeppen-geiger.vu-wien.ac.at), Poland has a humid continental climate and Portugal has a Mediterranean climate. Depending on the region, insolation in Portugal can be more than twice as intensive as in Poland, with the highest insolation levels observed in the south of Portugal. While the average temperatures in the warmest months may be similar in both countries, the average winter temperatures differ in 12 C, with negative values in Poland (À3.7 C) and above 3.0 C in Portugal. The local minimum temperature has been used to predict the survival of bacteria in specific environments, being higher temperatures responsible for enhanced survival or proliferation of bacteria (including pathogens and antibiotic resistant) (MacFadden et al., 2018). Also precipitation, which may occur in the form of rain or snow, presents higher average annual values in Poland than in Portugal (Table 1). Nevertheless, the highest precipitation periods occur in summer in Poland and in the winter in Portugal (Table 1).
Precipitation is one of the factors that may directly influence the volume of wastewater entering urban wastewater treatment plants (UWWTPs), when combined sewers are used, as is common in urbanized areas. This additional flow of water, mainly stormwater, may decrease the residence time of wastewater in the treatment tanks, therefore affecting the efficiency of the treatment, raising the risks that increased loads of ARB and ARGs are released to the environment (Eramo et al., 2017;Honda et al., 2020). Moreover, stormwater may be a source of contaminants such as heavy metals or microorganisms (Barbosa et al., 2012). Previous studies have shown that precipitation may influence the load of pathogenic bacteria and ARGs abundance, suggesting the relevance of this parameter to accurately monitor ARB/ARGs in aquatic environments (Ahmed et al., 2018;Di Cesare et al., 2017). Hence, the level of precipitation and the time of the year when it is more intense, may contribute to explain differences in the regional or seasonal pattern of antibiotic resistance occurrence. In turn, in UWWTPs with open tanks, solar radiation may also influence the treatment, contributing both to the photodegradation of chemical compounds such as antibiotic residues or bacteria photoinactivation (Rizzo et al., 2012). Besides, solar radiation may facilitate the application of advanced oxidation processes (AOPs), reducing the costs of advanced treatment processes (Marcelino et al., 2015). It can be argued that the relatively high temperatures observed in Mediterranean countries, such as Portugal, may contribute to improving removal efficiencies of the biological treatments (Zouboulis & Tolkou, 2015). In both analyzed countries, treated wastewater is mainly released into rivers (EEA, 2021). UWWTPs are most often built near a river downstream of served populations and may represent a source of chemical and microbial pollution of/in the receiving water body. The temperature of river water can affect microbial growth, whereas ultraviolet (UV) radiation might limit the growth of some microorganisms (Mishra et al., 2019). The impact that the discharge of treated wastewater may have on freshwater may be estimated considering the dilution factor per countrythe ratio between the volume of freshwater available and the domestic wastewater discharge (Keller et al., 2014). Although Poland has larger basin areas and longer rivers than Portugal, the estimated dilution factor for Poland (38.71) is 1.5 times lower than the one estimated for Portugal (61.23) ( Table 1). This may contribute to the lower percentage of bathing waters classified with excellent or good quality (measured based on the monitoring of E. coli and intestinal enterococci) -$90% compared to the 99% reported for Portugal (EEA, 2020b). The higher number of coastal bathing sites in Portugal also contributes to these numbers, as coastal bathing sites are generally better than inland bathing sites (EEA, 2020b). However, in some cases, wastewater discharges may be also contributing to the degradation of the bathing water quality. A well reported case was the Artur owek ponds located in the Northern area of Ł od z, in Poland (EEA, 2020a).
Although the gross domestic product per capita based on purchasing power parity (GDP per capita, PPP) is almost the same in Poland and Portugal (33 739.40 vs. 33 131.41, respectively), the investment in healthcare expenditure relative to GDP (%) is lower in Poland than in Portugal (Table 1). A negative and significant correlation between GDP per capita and antibiotic resistance prevalence has been demonstrated at the global level (Savoldi et al., 2020). In general, countries with high GDP per capita have improved conditions to prevent infections, which explains such correlation , although it can be argued that higher antibiotic consumption, might contribute to the antibiotic resistance propagation, in the absence of adequate sanitary or health care conditions (Collignon & Beggs, 2019). In Portugal, the business sector seems to be more active, with a higher birth rate of new companies, mainly dedicated to services ($76%). In Poland, companies are more evenly distributed between the services (57.4%) and industrial (40.2%) sectors (Table 1). The different types of companies present in the two countries may have a reflex in the type of residues that are being produced and discharged into UWWTPs and water bodies. The impact of some industries as sources of environmental contamination (e.g., heavy metals and antibiotics), may drive or accelerate the dissemination and possible diversification of antibiotic resistance in aquatic environments (Hubeny et al., 2021).
The indicators described in this section related to geography, demographics, climate, as well as to the economic status of Poland and Portugal are directly or indirectly connected to the volume of wastewater collected by sewage systems, and their contamination by chemical and microbial pollutants.

Urban wastewater treatment plants (UWWTPs)
Globally, a significant part of the human population lives in urban areas and an increasing percentage of the population is connected to the municipal sewage system. Therefore, it is assumed that UWWTP influents reflect the microbiome of the local human population, including the presence of ARB, ARGs, and mobile genetic elements (MGEs) (Hendriksen et al. 2019;P€ arn€ anen et al. 2019). UWWTPs commonly employ several operations, including mechanical, biological, and chemical processes. Comparing urban wastewater agglomerations larger than 2 000 population equivalent (p.e.) in 2016, Poland had 1674 and Portugal 472 ( Fig. 1), which generated a total load of 36 Â 10 6 m 3 and 12 Â 10 6 m 3 (EEA, 2021), respectively. These values are in good agreement with the population size in both countries, as it is also confirmed by Eurostat data (Eurostat, 2020) that indicates that 73.6% and 85.0% of the population is connected to UWWTP in Poland and Portugal, respectively. Conventional wastewater treatment may include distinct successive stages: primary, secondary, and tertiary (more stringent) treatment (Quach-Cu et al., 2018). The percentage and number of UWWTPs (for agglomerations ! 2 000 p.e.) using different stages of treatment in Poland and Portugal is presented in Fig. 1. According to the data available for the year 2016, in both countries most of the UWWTPs (97-98%) had at least secondary wastewater treatment system (Fig. 1). Only a small fraction of UWWTPs relied exclusively on primary treatment (Fig. 1), a method that is mostly used as a pretreatment for further secondary and more stringent treatments. As the sole treatment process, primary treatment systems are usually applied in small plants with low daily processing values. Preliminary treatment is not expected to eliminate microorganisms in the liquid stream, and the reduction of viral, bacterial, and protozoan pathogens has been described to reach 0 to 1 log units after conventional primary sedimentation. It makes UWWTPs based solely on this treatment potential sources for the spread of antibiotic resistance (Mar ın et al., 2015;Oakley, 2018;Szostkova et al., 2012).
Although the biggest share of the UWWTPs in both countries operates with a secondary treatment ($57-61%) ( Fig. 1), according to the latest EEA data (EEA, 2021) this type of treatment serves 14.0% and 46.7% of the population in Poland and Portugal, respectively. Worldwide, the most common biological treatment processes include: the activated sludge (AS), aerobic sequencing batch reactor (SBR), membrane biological reactor (MBR), biological filter (BF), and upflow anaerobic sludge blanket (UASB) Yuan et al., 2016). In Poland, the largest UWWTP (Czajka, Warsaw) with a dimension of 2 100 000 p.e. operates with an AS-based technology. The Portuguese largest UWWTPs are located in Lisbon and Loures, as Alcântara (756 000 p.e.) and Frielas (700 000 p.e.) UWWTP, operating with biological treatment carried out by BF using BIOSTYRV R technology and modified AS process (NeredaV R technology), respectively (ADP, 2018; MPWIK, 2021). On average, during biological treatment, the abundance of ARB and ARGs is efficiently reduced by 2 log-units . When comparing different methods of treatment: SBR and MBR processes significantly reduce ARB abundance, with log values ranging between 2.70-3.13 and 2.80-3.54, respectively, followed by AS system of 1.76-2.06 log More stringent treatments include disinfection (chlorination, UV, or ozonisation), sand filtration, microfiltration (e.g., membrane filtration), and other types of unspecified additional treatments. Source: The European Environment Agency (EEA). Available from: https://www.eea.europa.eu/ (Yuan et al., 2016). BF and UASB are assumed to have lower reduction levels. AS and SBR also demonstrate significant potential on ARGs reduction, with log values ranging 2.36-4.24 and 1.66-3.56, respectively (Yuan et al., 2016). BF resulted in a lower reduction of ARGs abundance (0.58-1.18 log-units). According to the same study (Yuan et al., 2016), MBR and UASB are the least effective in ARGs removal from wastewater. The AS treatment, the most common worldwide, has been suggested as being more effective than BF in removing ARB and ARGs (ARB log reduction: 1.76-2.06 and 2.36-4.24, respectively; ARGs log reduction: 0.87-1.23 and 0.58-1.18, respectively) (Yuan et al., 2016).
More stringent treatment, meaning postsecondary treatment, is applied to wastewater discharged in areas covering 76% of EU territory (EuropeanCommission, 2017). In Poland and Portugal, more stringent treatment is used to treat wastewater from 59.5% to 38.0% of the population, respectively (EEA, 2021). This type of treatment is utilized in $36% of the UWWTPs in Poland and $41% in Portugal (Fig. 1). More stringent treatment often includes disinfection, an important barrier to reduce the release of ARB and ARGs into the environment (Guo et al., 2013;Rizzo et al., 2020). However, in Poland, permanent disinfection of treated wastewater in properly operated UWWTP (mechanical and biological) is not used. According to Polish law on collective water supply and collective sewage disposal (Michałkiewicz et al., 2011;PolishLaw, 2005), only wastewater discharged from hospitals of infectious diseases, and those with such wards, and from blood donation stations must be disinfected before being discharged to municipal wastewater systems. Authorities' recommendations regarding the pretreatment of hospital effluents are not in place in Portugal. Nevertheless, in Portugal, disinfection is used in numerous UWWTPs, mostly in coastal regions and during the summer season. For example, six of the 15 largest Portuguese UWWTPs use UV disinfection process (Guia, Alcântara, Frielas, Barreiro/Moita, Ave, and Chelas treatment plants), sometimes supplemented by sand filtration (Guia, Alcântara, and Chelas) (UWWTD, 2016). As countries belonging to European Commission, Portugal and Poland are under the Council Directive 91/271/EEC (EuropeanComission, 2001) concerning urban wastewater treatment that was adopted on 21 May 1991 and that was under Online Public Consultation till July 2021. This Directive does not include any microbiological or specific chemical pollutant as surrogates to assess treatment quality, although each country is free to impose more restrictive criteria. This Directive aimed to minimize the impacts in the receiving environment, considering the type and load of anthropogenic substances, particles and microorganisms that might be a threat for the environment and humans 30 years ago. Both countries, Poland and Portugal, follow the European Directive.
In different world regions, the reuse of UWWTPs effluents has been encouraged as a method of water protection, although concerns regarding the spread of antibiotic resistance have been raised (Hong et al., 2018;Sorinolu et al., 2020;Zammit et al., 2020). In May 2020, the European Commission launched a new regulation (2020/741) on the minimum requirements for water reuse that defines the criteria for safe water reuse in agriculture irrigation (EU, 2020). The microbiological parameters include microorganisms of fecal origin and do not specifically address the antibiotic resistance issue. In Poland, the use of treated wastewater for agriculture is legally permitted (EuropeanCommission, 2016;PolishLaw, 2017). However the practice is not applied and the reuse of treated wastewater for irrigation in agriculture has not been reported (EuropeanCommission, 2016;Lemitor, 2019). In Portugal, around 1% of the treated wastewater was reused in 2019 (> 78 million m 3 /year) for agriculture and golf course irrigation, industry, or urban uses (ERSAR, 2021;Rebelo et al., 2020).

Antibiotics consumption
Antibiotics have been broadly used for years in humans, veterinary, livestock, and agricultural purposes. The high loads of antibiotic residues released in the environment represent an important fraction of contaminants of emerging concern (CECs), increasingly detected at low concentrations in wastewater, surface, ground, and drinking waters, as well as in soils and sediments (Booth et al., 2020;Rodriguez-Mozaz et al., 2020). Over the almost 80 years of antibiotic use, antibiotic residues, ARB, and ARGs have accumulated and spread in the environment, constituting a triad of presumably interlinked CECs. The use of antibiotics has been associated with resistance development, as numerous studies have demonstrated (Caron & Mousa, 2010;Llor & Bjerrum, 2014). This knowledge laid the foundations to encourage and recommend stewardship in antibiotic use, adopted by different European countries in the first decades of the 20 th century (Klein et al., 2018).
Poland is among the European countries with the highest antibiotic consumption (community and hospital sector combined), reaching values of 23.6 defined daily dose (DDD) per 1 000 inhabitants per day in the year 2019, when the maximum was 34.1 in Greece (ESAC-Net, 2020). In the same year, Portugal had an average consumption rate of 19.3 DDD per 1 000 inhabitants, close to the average level in EU/EEA countries. The average total consumption of antibacterials for systemic use (ATC group J01) in the EU/EEA was 19.4 DDD per 1 000 inhabitants, ranging from 9.5 to 34.1 (ESAC-Net, 2020). Poland and Portugal had managed differently the antibiotic stewardship recommendations, as the compound annual growth rate (CAGR) of total antibiotic consumption reveals. Between the years 2010 and 2019, the CAGR values were calculated to be 2.4% for Poland, corresponding to an increase of use, and À0.4% for Portugal, meaning a slight decrease. These trends were similar both in the community (primary care sector) and in the hospital sectors in each country (Fig. 2). In the community sector, during the same period, Poland had a continuous increase (average 20.9 DDD per 1 000 inhabitants), while in Portugal had a Source: European Center for Disease Prevention and Control. Antimicrobial consumption in the EU/EEA, annual epidemiological report for 2020 (ECDC, 2020) and ESAC-Net interactive database, available from: https://ecdc.europa.eu/en/antimicrobial-consumption/surveillance-and-disease-data/database slight decrease (average 17.6 DDD per 1 000 inhabitants), which followed the EU/EEA trend (average 19.0 DDD per 1 000 inhabitants). In the hospital sector, Poland and Portugal were below the average value for EU/EEA (1.8 DDD per 1 000 inhabitants, range 0.8 to 2.5), with values of 1.40-1.42 DDD per 1 000 inhabitants.
Beta-lactam antibiotics are the most used in the community and hospital sectors in EU/EEA, including Poland and Portugal (Fig. 3). In the community, the second most used class of antibiotics was macrolides, lincosamides and streptogramins (MLS), followed by tetracyclines in Poland, as well as in EU/EAA, contrasting with Portugal where the third class of most used antibiotics was quinolones. In the hospital sector, MLS was the second most used class of antibiotics in Portugal, contrasting with Poland where this position was occupied by quinolones, as well as in EU/EAA (Fig. 3).
Although antibiotics are mainly used in human health care, the prophylactic use of antibiotics in food animal production (e.g., poultry, swine, and cattle) takes the largest proportion of antibiotic consumption . Confirming this general European trend, data referring to the year 2014 showed that both in Poland and Portugal were used about double of antibiotics in animals, compared to humans, specifically 578 vs. 263 and 190 vs. 76, respectively (ECDC/EFSA/EMA, 2017). Just a very small fraction (< 1%) of the antibiotics used for animals are consumed by companion animals (Fig. S1a).
The latest ESVAC (European Surveillance of Veterinary Antimicrobial Consumption) report published in 2020, showed that sales of antibiotics for use in animals in Europe fell by more than 34% between 2011 and 2018 (EMA, 2020). Contrary to the European trend, in Poland, from 2011 to 2018, it was registered an increase of 33% in overall sales (mg/population correction unit (PCU)) of veterinary antimicrobial agents (EMA, 2020). In Portugal, in the same period, the overall sales (mg/PCU) increased 15%. The patterns of sales (mg/PCU) in Poland and Portugal were, in general similar, characterized by the predominance of tetracyclines, beta-lactams, and MLS (Fig. S1b). However, the existing differences may be influenced by the type of animal population, production systems, and prescription guidelines or habits in both countries (EMA, 2020).

Antibiotic resistance in hospital and primary care isolates
In 2019, 30 EU/EEA countries participated in the European Antimicrobial Resistance Surveillance Network (EARS-Net) with routine antimicrobial susceptibility testing (AST) data from invasive (blood or cerebrospinal fluid) isolates under surveillance: Escherichia coli (Fig. 4a), Klebsiella pneumoniae (Fig. 4b), Pseudomonas aeruginosa (Fig. 4c), Acinetobacter species (Fig. 4d), Streptococcus pneumoniae, Staphylococcus aureus (Fig. 4e), Enterococcus faecalis and Enterococcus faecium (Enterococcus species, Fig. 4f) (ESAC-Net, 2020). Portugal reported an estimated population coverage of 97% with high sample representativeness (ECDC, 2020). Poland is one of the few countries reporting medium geographical coverage or hospital data representativeness, with a comparatively low population coverage (17%) of EARS-Net contributing laboratories and hospitals. This is an important limitation to the comparison between countries, with unknown implications in the reliability of data analysis and interpretation. However, it is suggested that in general, higher percentage of resistance was observed in Poland than in Portugal and EU/EEA (population-weighted mean percentage) (Fig. 4).
In the group of E. coli isolates, the highest resistance prevalence values (%) were reported for aminopenicillins (61.6% in Poland and 58.5% in Portugal) and fluoroquinolones (33.0% in Poland and 26.5% in Portugal), followed by resistance to third-generation cephalosporins and aminoglycosides (Fig. 4a). Combined resistance to fluoroquinolones, third-generation cephalosporins and aminoglycosides in K. pneumoniae isolates was reported at a higher percentage for both Poland (45.0%) and Portugal (26.5%) countries than the EU/EEA average values (Fig. 4b). P. aeruginosa, with high rates of fluoroquinolone resistance, presented higher carbapenem-resistance Source: European Center for Disease Prevention and Control. Antimicrobial consumption in the EU/EEA, annual epidemiological report for 2020 (ECDC, 2020) and ESAC-Net interactive database, available from: https://ecdc.europa.eu/en/antimicrobial-consumption/surveillance-and-disease-data/database values than E. coli or K. pneumoniae, and a higher percentage in Poland (24.4%) than in Portugal (17.8%). Acinetobacter species displayed the major differences in resistance prevalence between Poland and Portugal (39.9% for carbapenem and 59.4% for fluoroquinolones). In this bacterial genus, the percentage of fluoroquinolone resistance reported was 85.5% in Poland and 26.1% in Portugal, while for carbapenems it was 71.0% in Poland and 31.1% in Portugal (Fig. 4d). In Portugal, the percentage of methicillin-resistant Staphylococcus aureus (MRSA) was 34.8%, a result of a significantly decreasing trend between 2015 and 2019 (ECDC, 2020). In the same period, the MRSA prevalence reported by Poland ranged 15-16% (ECDC, 2020). Since 2016, vancomycin resistance prevalence in Enterococcus species (E. faecalis and E. faecium) was low in Portugal, while it increased in Poland from 17.7% in 2015 to 44.0% in 2019 (ECDC, 2020). High-level gentamicin resistance in E. faecium decreased over 10 years of surveillance in both countries, from 64.9% to 46.3% in Poland and 53.3% to 21.8% in Portugal. This resistance phenotype also decreased in Portugal in E. faecalis for the period 2015-2019 (ECDC, 2020).

Antibiotics in wastewater
Wastewater has been considered an important mirror of the community use of antibiotics and antibiotic resistance status (Hendriksen et al. 2019;P€ arn€ anen et al. 2019;Rodriguez-Mozaz et al., 2020). This section and the following focus on this aspect. Antibiotics are usually only partly metabolized in the human body, being excreted in urine and feces as active compounds, with the possibility of reaching the municipal wastewater system in this form. Current UWWTP technologies, usually based on AS process, have been developed and successfully applied to control the dissemination of organic matter and nutrients (mainly nitrogen and phosphorus). Antibiotics may be refractory to biodegradation in conventional UWWTPs (Michael et al., 2013;Zhang & Li, 2011). According to ESAC-Net data, in Poland higher consumption of antibacterials was noted than in Portugal, especially at the community (primary care sector) level (Figs. 2 and 3). This tendency is however not mirrored in the concentration of antimicrobials observed in wastewater, as a higher concentration of antimicrobial agents was detected in Portuguese than in Polish wastewater systems (Table 2). However, the data being compared result from sporadic sampling events and possible influencing variables (e.g., season, share of hospitals and nursing/residential care facilities, retention time in the sewer network, wastewater treatment systems and sewage sludge management, among others) are not being considered. Other reasons limit a systematic comparison, mainly the fact that the measurement of antimicrobial agents concentration in wastewater is mainly performed as academic research and the data is scattered and rare. For example, data of beta-lactams quantification in wastewater is available only for Portugal (Table 2). Even when data are available for both countries, factors such as the number of studies/UWWTPs that were sampled, the type of sample that was collected (grab or composite), or the season in which samples were collected may have interfered with the results. Langas et al. (2019), observed high seasonal variability in the quantity of some targeted antibiotics in the raw wastewater of 4 Polish UWWTPs. In general, the concentration of antimicrobials in the raw wastewater was lower in summer than in winter (e.g., azithromycinup to 0.216 mg/L and up to 24.145 mg/L; clarithromycinup to 2.166 mg/L and up to 7.294 mg/L; sulfamethoxazoleup to 0.387 mg/L and up to 2.020 mg/L, respectively) (Langas et al., 2019). This variability can be explained by the increasing consumption of these antibiotics due to a higher incidence of respiratory tract infections in the winter (Lange et al., 2020).
In many cases, antimicrobial agents were detected in the UWWTP influent, both from Poland and Portugal, in concentrations higher than the predicted no effect concentrations (PNECs) for resistance selection, as was proposed by Bengtsson-Palme and Larsson (2016). A similar tendency was also noted for treated wastewater since conventional UWWTPs based on secondary treatment (which account for over 50% of all facilities; Fig. 1) remove antimicrobial agents with different efficiency (Table 2). It is also important to note that some antimicrobials (e.g., penicillins) are degraded easier than, e.g., fluoroquinolones or tetracyclines, which may accumulate in treatment sludges or in the water body receiving the treated wastewater. In Poland, tetracycline, which use is mainly in animals as reported above, was detected in treated wastewater in concentrations up to 0.240 mg/L, while in Portugal it varied from undetected to 2.420 mg/L.
The results obtained for macrolides in raw wastewater indirectly confirmed the growing use of the newer generation (i.e., broad-spectrum) of antimicrobials in Poland, such as the new class of macrolides (azithromycin and clarithromycin, if compared with erythromycin) (Table 2). However, such tendency was not confirmed in Portugal, where in the UWWTP influent was mainly detected erythromycin (up to 2.300 mg/L). It can be partly explained by the difference in medication structure in both countries, as well as the possibility to buy antimicrobials over the counter. It was estimated, for example, that in a UWWTP serving a municipality of about 571 350 residents (average flow 93 000 m 3 /d; average load 750 000 p.e.) the mean load of azithromycin in the UWWTP inlet might reach 265 kg per year, and be reduced to 76.1 kg per year in the outlet (Langas et al., 2019). Azithromycin is a re-positioned macrolide antibiotic, active against both Gram-positive and -negative bacteria. However, the unique chemical architecture of this and other macrolides makes that in addition to the antibacterial effect they may also have an antiviral effect. Thus, the potential to treat or prevent viral co-infection (including new influenza A(H1N1), Zika, and possibly others) (effectiveness against SARS-CoV-2 is under debate) (Sterenczak et al., 2020;Tran et al., 2019), might increase their usage.
From the above, it can be concluded that constant discharge of treated wastewater constitutes an important load of pharmaceuticals introduced into the receiving water bodies, which relevant environmental burden must be assessed and mitigated whenever possible. Although there is a need to monitor some antimicrobials (e.g., amoxicillin, ciprofloxacin, and macrolides: n.d., not detected; n.a., not available. PNECs -predicted no effect concentrations for resistance selection provided by Bengtsson-Palme and Larsson (2016). erythromycin, clarithromycin, and azithromycin) and other micropollutants, such as azole compounds, at a European level (Decision_2020/1161,1161 2020), there are no requirements regarding the extent at which they must be removed from wastewater. Efforts to remove micropollutants from wastewaters have been under discussion and an example of implementation and milestone definition is illustrated by Switzerland, where in 2016 was launched the legal basis for an additional (fourth) step of wastewater treatment. The goal is that by 2040 treatment processes are capable of reducing in 80% (primary clarified wastewater vs. final effluent) different surrogate substances (minimum 6 out of 12; clarithromycinrepresenting antibiotics; amisulpride, citalopram, venlafaxineantidepressants; irbesartan, hydrochlorothiazideantihypertensives; diclofenacanti-inflammatories; metoprololbeta blockers; carbamazepine, candesartantranquillisers; and benzotriazole, mecopropother substances). Special concern was given to populated regions, where surface water resources serve as a source of potable water as well as treated wastewater receivers have limited dilution capacity. The feasibility of upgrading (selected) UWWTPs to more advanced treatment, capable to eliminate a broad range of micropollutants at reasonable costs (in fit-for purpose manner) deserves more investigation.

ARB and ARGs abundance in wastewater
UWWTPs are recognized reservoirs of antibiotic resistance, where it is expected to find a high relative abundance of ARB and ARGs. Culture-dependent methods are commonly used to study the abundance of ARB, while ARGs quantification is frequently based on real-time PCR . These have been the methods used for screening antibiotic resistance in raw and treated wastewater in Poland and Portugal. Because both are targeted methods and the experiments were designed independently, the comparison of data may be biased by the monitoring of distinct bacterial groups and ARGs (Tables S1 and S2). As noted before (Berendonk et al., 2015), the inexistence of recommendations about monitoring targets and methods is a major limitation to assess impacts and control risks due to environmental antibiotic resistance. However, the volume of studies on the subject suggests that some interstudy comparisons provide interesting insights (Krzeminski et al., 2019;Manaia et al., 2016). Hence, even considering that data from Portugal frequently refers to the broad group of enterobacteria, while Poland data refer to coliforms or Escherichia coli, it is possible to infer major patterns in both countries (Table S1). An interesting observation is that despite the variations observed over distinct sites or sampling campaigns, Polish UWWTPs seem to achieve higher reductions of cultivable ARB. In both countries, total heterotrophic ARB were generally above 4 log-units colony forming units (CFU)/ mL in raw wastewater, being the treatment processes responsible for reductions of 2-3 log-units in Poland and of 1-2 log-units in Portugal (Table S1, Fig. 5a). Although loads of amoxicillin resistant bacteria were higher in Poland influents, due to the higher removal rates, after treatment the abundance of these culturable bacteria was identical in both countries (3-5 log CFU/mL). Enterobacteria presented higher counts in Portuguese wastewater, both before and after the treatment. However, these results may have been biased by the fact that coliforms and E. coli, measured in Poland, are subgroups of enterobacteria (Table S1). Marano et al. (2020) used a harmonized methodology to enumerate cefotaxime resistant coliforms in the influent and effluent of 57 UWWTPs, located in Europe, Asia, Africa, Australia, and North America (a total of 22 countries), including Poland and Portugal. In that study, Portugal presented lower counts of cefotaxime resistant coliforms in raw wastewater (1 log-unit lower) than Poland, although also lower removal efficiencies (removing 1-2 log-units, while in Poland were removed 1-3 log-units). Enterococci presented counts ranging 3-4 log CFU/mL in the influents of both countries, although higher loads in the final effluents in Portugal (1-2 log-units CFU/mL in Poland vs. 1-3 log-units CFU/mL in Portugal). In contrast, vancomycin resistant enterococci presented higher counts in influent and effluent samples in Poland (1-2 log-units above the values observed for Portugal) (Table S1, Fig. 5a). This observation agrees with what was observed at the clinical level (Fig. 4F), with a higher percentage of vancomycin resistant enterococci in Poland than in Portugal (17.7% in Poland vs. 3.0% in Portugal). The relationship between clinical and wastewater vancomycin resistant enterococci in Poland was suggested by Sadowy and Luczkiewicz (2014). These authors found isolates belonging to the nosocomial HiRECC (high-risk enterococcal clonal complex) in raw and treated wastewater as well as in the receiving coastal area of Gdansk Bay, Poland, and had evidence that suggested the ability of those clones to survive in the environment. These observations are aligned with the perspective that the urban community resistome is mirrored in their sewage (Aarestrup & Woolhouse, 2020;P€ arn€ anen et al. 2019). In turn, the contrast observed between Poland and Portugal suggests that local conditions may shape the fate of clinical bacteria thriving in the environment, as has been discussed about the ecology of enterococci from different geographic regions (Blanch et al., 2003).
Because most wastewater bacteria are non-culturable, the measurement of genes brings additional and relevant information about treatment efficiency. Genes conferring resistance to betalactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, and integrase genes associated with integrons were among the most frequently monitored in Poland and Portugal (Table S2). Wastewater treatment unequivocally contributes to reducing the absolute gene abundance (expressed per volume of wastewater), although if a specific gene suffers lower removal rates than the average of bacteria, its relative abundance (expressed per 16S rRNA gene) may increase, suggesting the enrichment of that gene (Makowska, 2019;Makowska et al., 2016;Narciso-da-Rocha et al., 2014;Osi nska et al., 2020;Rocha et al., 2019;Zieli nski et al., 2020). Figure 5. ARB abundance (a), ARGs abundance (b) and relative abundance (c) in UWWTP influent (I) and effluent (E), in Poland (PL) and Portugal (PT). R AMX , resistant to amoxicillin; R TET , resistant to tetracycline; R CIP , resistant to ciprofloxacin; R CTX , resistant to cefotaxime; R VAN , resistant to vancomycin. bla OXA includes bla OXA-A , and bla OXA-1 . Note: Effluents from tertiary treatment are not represented in this figure; data is available in Tables S1 and S2. Most of the Polish data on Enterobacteria is reporting coliforms of E. coli counts (please see Table S1 for detail).
The data compiled in Table S2 shows that treatment promoted reductions of total bacteria, measured based on the 16S rRNA gene quantification, of up to 1 log-unit (gene copy/mL) in both countries. This suggests that the culturable bacterial groups examined are more efficiently removed during wastewater treatment (2-3 log-units for Poland and 1-2 log-units for Portugal) than total bacteria, and/or that DNA-based methods may overestimate inactive bacteria (Tables S1 and S2, Fig. 5a and b). Contrary to what was observed for ARB, the removal of ARGs was in the same order of magnitude in both countries (frequently 1-2 log-units per volume of water). Probably as a result of the different endurance of the host bacterial groups, treatment may lead to distinct patterns of variation in different genes. While in general there was a decrease in the abundance (per volume), the relative abundance (normalized per 16S rRNA) did not vary after treatment, although in some occasions it decreased or increased (Table S2, Fig. 5c). This general pattern is expected since ARGs hosts and total bacteria are, in principle, removed at the same rate, with some exceptions for specific bacterial groups that are more or less extensively removed during treatment. Different studies have explored these dynamics by monitoring ARGs and the composition and structure of the wastewater bacterial communities in parallel Grehs et al., 2019;Lira et al., 2020;Narciso-da-Rocha et al., 2018). Another important driver to explain the variable behavior of ARGs during wastewater treatment may be the accumulation of the bacterial ARGs hosts in activated sludges (Meng et al., 2020).
Concerning beta-lactamase genes (bla TEM , bla OXA , bla SHV , bla CTX-M , and bla VIM ), bla TEM and bla OXA were the most abundant in the wastewater of both countries. Although the abundance of those two genes varied over wastewater samples in Poland (Fig. 5b), in general decreased 1-2 log-units (per volume) after the treatment, while the relative abundance was fairly stable. In Portugal, the decrease in bla TEM reached 3 log-units, higher than of the 16S rRNA gene, as was reflected also in the decrease of the gene relative abundance. The opposite was observed with bla OXA that might have been occasionally enriched after treatment (Fig. 5b,c). Also, the genes bla CTX-M and bla VIM were not efficiently removed from Portuguese wastewater. In Poland, the removal of the genes bla CTX-M , and bla VIM was higher, although only occasionally with implications on the reduction of the relative abundance (Fig. 5c). The gene bla SHV presented higher removal in Portuguese than in Polish UWWTPs. The differences observed for the removal of these ARGs, often associated with enterobacteria, may be connected to the ecology of bacteria, and the higher stability of some bacterial groups (heterotrophs or enterobacteria) in Portugal than in Poland. These effects may need to be considered when additional control measures are designed.
The abundance of the tetracycline resistance gene tetM in raw wastewater from Poland ranged 2-6 log-units/mL, with reductions of 1-3 log-units after the treatment. Only one study quantified tetM in Portugal, being quantified 5 log-units/mL of treated wastewater, the highest value detected in equivalent samples from Poland (Fig. 5b). The fluoroquinolone resistance gene qnrS occurred in similar abundance in Polish and Portuguese raw wastewater, being suggested a more efficient removal in Portugal, reaching 3 log-units (per volume) vs. 1 log-unit observed in Poland (Fig. 5b). The sulfonamide resistance genes sul1 and sul2, and the integrase intI1 gene were observed to be weakly removed by the wastewater treatments used in both countries, with maximum removal of 1 log-unit in the genes abundance and no change in the relative abundance (Table S2, Fig. 5b,c). The abundance of sul1 and intI1 was observed to be in the same order of magnitude in Poland and Portugal, although sul2 was more abundant in both Portuguese influents and effluents.

Does the context matter?
One of the problems of dealing with the monitoring of dynamic and complex systems as UWWTPs is the high variability due to external and often not measurable variables. The values of ARB and ARGs abundance presented in this study reflect such variability that may be due to the properties of the wastewater, the treatment processes, analytical methodological options, season of sampling campaigns, among others. Studies conducted with multiple UWWTPs have highlighted that a vast array of factors may influence both the resistance in the raw wastewater and the treatment efficiency. If these differences have implications on the final impact that UWWTPs have in the receiving environment or if they can be explained based on measurable and controllable variables is still a major question Limayem et al., 2019). One of the outcomes of the data compilation herein presented is the evidence that a holistic vision is necessary to better understand and control antibiotic resistance dissemination. It is not only important to measure the abundance of ARB and ARGs that are being released by the UWWTPs to the environment, but also to understand their ecology in a broader context, as well as the context where it occurs and the respective permissiveness to ARB dissemination. This knowledge may provide useful guidelines for optimizing the treatment systems regarding antibiotic resistance control. The characterization of the antibiotic resistance genotypes of some bacterial isolates recovered from wastewater samples has been performed both in Poland and Portugal (Table S3). Environmental E. coli and Aeromonas spp. are important harbors of a high diversity of ARGs, namely conferring resistance to beta-lactams and quinolones, and some studies have shown that some of those strains can survive treatment, even when UV or ozone disinfection is used (Dolejska et al., 2011;Tavares et al., 2020;Varela et al., 2015;. Enterococcus faecium, Klebsiella pneumoniae, and Enterobacter spp. are other important vehicles of antibiotic resistance dissemination in wastewater (Table S3). Although the characterization of isolates is very important for better assessing the association of ARGs to their hosts, it has the limitation of requiring cultivation disregarding the non-cultivable bacteria fraction, which are the majority in environmental samples . The use of culture-independent approaches surpasses these limitation and can be applied using non-targeted methods to analyze the wastewater resistome. The use of correlation and clustering analysis allowed to infer for Portuguese wastewater which taxonomic groups are potential harbors of ARGs. For example, Fernandes et al. (2019) concluded that members of the families Aeromonadaceae, Campylobacteraceae, Veillonellaceae, [Weeksellaceae], and Porphyromonadaceae were positively correlated with the genes bla CTX-M , bla OXA-A , bla SHV , and intI1, while Intrasporangiaceae were negatively correlated. Narciso-da-  also observed that the families Campylobacteraceae, Comamonadaceae, Aeromonadaceae, Moraxellaceae, and Bacteroidaceae, highly abundant in raw wastewater samples, strongly correlated with the genes qnrS, bla CTX-M , bla OXA-A , bla TEM , bla SHV , sul1, sul2, and intI1.

Conclusions/final remarks
Poland and Portugal have similar demographic characteristics with different climatic conditions, resulting from the respective geographic location. Poland holds a strong industrial sector, while in Portugal services, mainly tourism, are pillars of the economy. Inevitably, this profile generates a distinct pattern and load of pollution in both countries. Unfortunately, these data are not available. While in Poland the consumption of antibiotics is higher, mainly at the community level, it is also in this country that health expenditure is lower, although legislation recommends dedicated treatment of hospital effluents generated at infectious diseases wards and blood donation stations. The fact that Poland has a poor adherence to clinical ARB surveillance (17% population coverage) and a later start of reporting antibiotic consumption explains why resistance trends are not yet to decrease as it is apparent in Portugal. This is both a cause and a consequence of a lower awareness for the problem of resistance in Poland than in Portugal, among the community and the authorities. Although Poland has a National Antibiotic Protection Program (http://antybiotyki.edu.pl/) in place since 2004. In Portugal, the national plan to fight antimicrobial resistance with the major objective of reducing the consumption of antibiotics (https://www.dgs.pt/ programa-nacional-de-controlo-da-infeccao) is in place since 2010 and is now producing the first results. After some years of a critical situation of clinical antibiotic resistance, Portugal is now tracing a corrective path characterized by the decrease of antibiotic use and also by the reduction of some pathogens, as MRSA. However, these measures may have a lower impact than in other world regions since wastewater loads of antibiotic resistance and removal rates due to treatment are in general low in this country. While wastewater resistome has been considered a good sensor of the resistance occurrence in a region (Aarestrup & Woolhouse, 2020;P€ arn€ anen et al. 2019), the treatment efficiency will be crucial to determine the impacts of community and health-care related resistance on the environment. UWWTPs are major barriers to protect the environment. Even if the situation improves at the clinical level, efforts are still needed to avoid the dissemination to the environment and, eventually, back to humans. In a European study, the comparatively lower capacity of UWWTPs to remove ARGs in Southern countries (where Portugal was included) compared with Northern countries was demonstrated (P€ arn€ anen et al. 2019), reinforcing the need for holistic characterization and interventions. Southern countries have higher temperatures, more favorable for human and animal commensal survival and proliferation in the environment, explaining positive correlations between antibiotic resistance and temperature (McGough et al., 2020). This trend was confirmed here mainly for culturable ARB. This also suggests that wastewater treatment may need to be customized for each socioeconomic and climate context. The load of resistance and microbial community composition of the sewage, the type of treatment implemented, the degree of general pollution, the organic matter load, and other factors such as temperature may dictate the efficiency of each treatment process. The duration of aeration/anoxic cycles, the addition of flocculation agents, the extension or reduction of hydraulic residence time, or the composition of treating activated sludge biomass may need to be adjusted to specific treatment scenarios, which are nowadays mainly conducted as black boxes in what concerns antibiotic resistance removal.
This study based on two countries aims also to demonstrate that, despite the numerous limitations, there is nowadays a plethora of antibiotic resistance information available worldwide that can be crossed with socioeconomic and climate data, allowing the establishment of informing principles to control antibiotic resistance.

Declaration of interest
No potential conflict of interest was reported by the authors.