In this study, Enterococcus spp. was used to evaluate the impact posed by treated wastewater of two local WWTPs on their receiver, the costal waters of Gulf of Gdansk. Total counts of enterococci observed for raw (W-INF) and treated (W-EFF, D-EFF) wastewater are consistent with our previous study, showing similar, over 99% reduction capacity of applied treatment processes. It is also reported in the literature that wastewater treatment processes based on activated sludge, although they focus on removal of nutrients and organic matter, simultaneously reduce faecal indicators with high efficiency, reaching up to 99.9% [18, 22]. Due to the high number of enterococci in raw wastewater, they are nevertheless released from a WWTP in a number up to 6.1x105 CFU per 100 mL. Thus, marine water sampled in the area directly impacted by treated wastewater (W-MOut and D-MOut) showed a higher number of enterococci than the one observed in Vistula River mouth, however, lower by four orders of magnitude in comparison to corresponding treated wastewater (W-EFF and D-EFF). It should be noted that in marine outfalls (W-MOut, D-MOut) as well as in Vistula River mouth enterococci were detected in number lower than 100 CFU per 100 mL, i.e. the value set out by New Bathing Water Directive 2006/7/EC for the costal water of “excellent quality” .
It is suggested that enterococcal species composition in faecal-impacted environment depends from the source of contamination. In this study, two species most commonly colonizing humans, E. faecalis and E. faecium comprised together from 73% to 88% of enterococcal isolates in tested wastewater samples (W-INF, W-BR, W-EFF and D-EFF), 73% and 61% in marine outflows (W-MOut and D-MOut, respectively) and 45% in the Vistula River mouth. The predominant species in wastewater and marine water impacted by treated wastewater was E. faecium (from 56 to 65%), which stayed at a similar level during treatment processes, as determined also in our previous study . Species distribution in wastewater samples is not well understood, and affecting factors may include differences in diet, climate, season and methods of detection. E. feacalis was found to be the most prevalent species in wastewater samples in Sweden , E. faecium in Spain, UK, Canada, France and Switzerland [26–29], while E. hirae predominated in Portugal and United States [22, 30]. Although E. faecalis is typically the most abundant colonizer of humans in the community (e.g. [26, 31]), E. faecium is generally most often associated with human microbial contamination [27, 29]. E. faecalis was reported from agriculture-impacted waters  and E. hirae likely originates mainly from cattle and pig faecal contamination , which explains high relative proportion of these two species in Vistula River mouth. Apart from the variability of contamination sources, proportion of particular species may differ due to the differences of ability to sustain environmental stresses. In this study, enterococcal composition might have been influenced by wastewater treatment processes and marine water characteristics.
The effect of wastewater treatment and marine environment was seen on phenotypes of antimicrobial resistance. The positive selection of bacteria with resistance patterns has been already suggested and noted in wastewater processes [19, 20, 22]. In this study, the resistance rates to ampicillin, ciprofloxacin, levofloxacin and erythromycin, noted for E. faecium from raw wastewater (W-INF) were lower than these observed for this species in the corresponding treated wastewater (W-EFF), however, not statistically significant (p > 0.05). Moreover, resistance rates to ampicillin, ciprofloxacin, tetracycline and erythromycin observed in marine outflow of WWTP Gdansk-Wschod (W-MOut) were higher than in treated wastewater of this plant. Such phenomenon was not observed for treated wastewater of WWTP Gdynia-Debogorze (D-EFF) and its marine outfall (D-MOut). Resistance rates among E. faecium originating from D-MOut were, in general, comparable to these detected in the Vistula River mouth. It should be noted that treated wastewater from WWTP Gdansk-Wschod has been discharged via marine outfall (W-MOut) for last 12 years while marine outfall of WWTP Gdynia-Debogorze (D-MOut) has been operating for last two years. The observed differences in resistance rate corresponding to the local impact caused by long and short-term operated marine outfalls (W-MOut and D-MOut) need further attention.
Significant rates of antimicrobial resistance, observed for isolates of E. faecalis from wastewater and marine/river waters, impacted by wastewater, are in agreement with a recent study performed in six European countries, showing that in Poland the rates of resistance for human isolates of this species were usually among the highest, both in the community and hospitals . To our knowledge, no corresponding data are available for E. faecium colonization in the community in Poland but a study focused on hospital VRE isolates showed their ubiquitous MDR phenotype and a high load of resistance genes . In our study no vancomycin resistance were detected, likely due to relatively low (below 10%) prevalence of VRE in Polish hospitals , although such isolates are isolated from the environment in other countries e.g., .
This study shows that hospital clones of E. faecium are important carriers of resistance genes in wastewaters. Most likely, in a significant part they originated from hospital wastewater treated together with communal wastewater. In our estimation, hospital wastewater constitutes approximately 0.2% of both WWTPs’ daily inflow. The release of hospital-associated clones of E. faecium to the environment was observed also by others [24, 28, 35, 36]. In our study, isolates belonging to the major nosocomial HiRECC (formerly named CC17) constituted altogether 24.6% of all isolates of E. faecium subjected to typing, and were present at all sampling sites. These isolates, apart from their inclusion into the HiRECC by MLST, demonstrated several other phenotypic and genotypic features typical for nosocomial E. faecium, i.e. they were resistant to ciprofloxacin and ampicillin, in vast majority showed MDR phenotype, and carried several resistance determinants, associated both with mutations of chromosomal genes (gyrA, parC, pbp5) and, in the case of aminoglycoside and tetracycline resistance, with gene acquisition. Plasmids likely played an important role in the later process, as some plasmid genes, such as rep2
pRUM and rep18
pEF418, and TAS genes axe-txe and ω-ε-ζ were significantly more abundant in isolates associated with HiRECC than among the remaining isolates. As yet, the knowledge of distribution of plasmid-associated genes in populations of E. faecium, especially in the context of their clonal composition, remains limited. A study on 93 isolates of different geographical origins and from various sites showed an over-representation of rep17
pRUM and rep
pMG1 among nosocomial E. faecium, while, in contrast to our observations, rep18
pEF418 was not detected . Moreover, we did not observe rep
pMG1 among HiRECC of E. faecium. These differences may be due to a high prevalence of VRE (58 isolates) among 93 isolates studied by Rosvoll et al. what may explain differences in the plasmid content. The same study also reported the co-localization of ω-ε-ζ with rep2
pRE25 and axe-txe with rep17
pRUM on the same plasmids, in concordance with our findings, showing a frequent joint occurrence of these pairs of genes. Another study, performed on 99 invasive isolates from Norway, mostly of HLGR phenotype, showed a significantly higher prevalence of rep2
pRE25 and rep17
pRUM among the major nosocomial HiRECC . This study as well some others [24, 39] also demonstrated a common presence of rep
pLG1 among E. faecium from hospital settings and in animals. Our findings indicate that this type of replicon is even more ubiquitous in E. faecium than previously reported. All rep genes mentioned above are typically associated with plasmids carrying various antimicrobial resistance genes [39, 40], GenBank accession number AF408195. In addition to genes determining antimicrobial resistance and genes associated with MGE, isolates of nosocomial HiRECC analyzed in our study carried such molecular markers as IS16 and the esp gene together with the intA gene characteristic for ICEEfm[41, 42]. Another pathogenicity factor, the fms19 gene of the pilin gene cluster 4 (PGC4) showed increased prevalence among these isolates. Similar observation was made for a representative collection of 433 E. faecium isolates from various sources .
In a study that followed enterococci in a continuum from hospital and retirement home wastewaters through a WWTP to a river, a gradual decrease in the proportion of E. faecium isolates representing hospital-associated STs was observed, likely due to the “dilution” of HiRECC representatives by more diverse and less drug-resistant strains originating from the community . Because in our study only selected representatives of obtained isolates were investigated, it is not possible to determine precise tendencies in their frequency from the WWTP influent to effluent and water, however, resistant enterococci, both HiRECC and non-HiRECC, are clearly able to survive the treatment process, what results in their release into marine and river waters. Moreover, using an additional typing method, MLVA, we were able to find isolates with the same characteristics in the effluent of WWTP and its marine outfall, suggesting ability of such clones to survive in the environment for at least some time. It seems plausible that hospital resistant clones accumulate several determinants that may promote their survival not only in the hospital settings but also in the environment. Such features would include resistance to disinfectants and ultraviolet light, additional metabolic pathways and ability to form biofilms. Moreover, determinants of some of the mentioned adaptation factors are found co-resident in plasmids harboring resistance genes, suggesting co-selection. For example, pLG1-type plasmids of E. faecium carry not only resistance determinants against aminoglycosides, glycopeptides and macrolides but also pili genes and carbon uptake-utilization genes [38, 39]. Pheromone-responsive plasmids of E. faecalis apart from resistance to aminoglycosides, glycopeptides, penicillins and macrolides may encode bacteriocins, additional pili and UV-resistance determinants . Thus, nosocomial HiRECCs, released by WWTPs, are equipped in whole sets of genes with various adaptive benefits and often located within MGE. It was also demonstrated that MGEs can be transferred in a WWTP , further underling the importance of threat posed by such organisms. There is a selection pressure for survival of resistant strains and their tranconjugants due to release of antibiotics to the environment, especially in the hospital effluents , and even sub-inhibitory concentrations may be effective in selection for resistance .