KPC-Producing Enterobacterales from Douro River, Portugal—Persistent Environmental Contamination by Putative Healthcare Settings

Carbapenemase-producing Enterobacterales (CPE) are a growing concern, representing a major public health threat to humans, especially in healthcare settings. In the present study, we evaluated the persistent contamination by carbapenem-resistant Enterobacterales in water from Douro River, Portugal. KPC-producing Enterobacterales were detected in five water samples separated chronologically by 15 days each. Susceptibility testing was performed by disk-diffusion-method according to Clinical and Laboratory Standards Institute (CLSI), phenotypic carbapenemase activity was evaluated by carbapenem inactivation method, presumptive identification of the isolates was performed by CHROMagar orientation and confirmed by API-20E. Carbapenemase genes were screened by PCR and the clonality of all isolates was assessed by XbaI-Pulsed Field Gel Electrophoresis (PFGE). Fifteen KPC-producing Enterobacterales isolates were selected, identified as multidrug-resistant and showed a resistance profile to non-beta-lactam antibiotics: sulfamethoxazole + trimethoprim (7/15), ciprofloxacin (3/15), fosfomycin (3/15) and chloramphenicol (2/15). Isolates were identified as (6) Escherichia coli and (9) Klebsiella pneumoniae. Our results suggest a punctual contamination with KPC-producing Enterobacterales continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. This work provides a better understanding on the impacts of water pollution resulting from human activities on aquatic environments.


Introduction
The emergence of carbapenemase-producing Enterobacterales (CPE) has been identified in recent years as one of the major public-health concerns over the last decade. Carbapenems constitute the last-resort class of antibiotics for nosocomial infections, as in some cases of infection there are very limited treatment options available, being associated with mortality rates that usually exceed 40% [1][2][3][4]. CPE are no longer restricted to healthcare settings, being detected in different ecological niches, such as river waters [5].
In Portugal, KPC prevails over other carbapenemases, with KPC-2 being the most prevalent in northern Portugal and KPC-3 being more associated with healthcare settings [6][7][8].
In 2015, as result of multidrug-resistant pathogens, particularly Klebsiella pneumoniae carbapenemase-producing Enterobacterales (KPC-E), approximately 33,000 people died in Europe. Nowadays, KPC-E is a concern due to the high level of endemicity observed in various areas worldwide, being considered as an urgent threat and one of the biggest global public-health challenges [9].
It is known that there are several connections between the environmental, human and animal compartments that allows for the movement of bacteria, as well as mobile genetic elements and antibiotic resistance [10]. The aquatic environment is often exposed to discharges from various sources, through inadequate handling in agriculture, healthcare settings and industry, collecting biological and chemical contaminants. This allows this ecosystem to perform as a vehicle for antibiotic-resistant bacteria and antibiotic resistance genes that circulate and spread in the environment, reaching and affecting humans [11,12]. It is worth noting that river ecosystems are very relevant, playing key roles in ecosystem functions such as drinking water, food, agriculture, animal production and leisure activities [13].
Presently, there are few epidemiologic studies regarding CPE recovered from rivers. The aim of this study was to explore the presence of CPE in the ecosystem of Douro river, an important river in the Porto metropolitan area and hence with a considerable anthropogenic impact and scarce contribution of animal faecal contaminants.

Carbapenemases and ESBL
Fifteen isolates producing carbapenemases were confirmed by the carbapenem inactivation method (CIM) and the carbapenemase phenotype was due to the presence of bla KPC genes (15/15). No other carbapenemase genes were detected.
More, all of the isolates additionally carried 1 to 3 of the most prevalent genes of ESBL-producing bacteria. Seven isolates carried bla CTX-M group 2 (7/15, 46.7%), bla TEM was detected in six isolates (6/15, 40%) and bla OXA in four isolates (4/15, 26.7%). Both bla CTX-M group 9 and bla SHV were not detected in any of the isolates.

Clonal Relation of KPC-Producing Enterobacterales
No clonal relation was detected in the E. coli or in the K. pneumoniae evaluated ( Figure S1). With exception of 3 E. coli isolates with similar pattern, which were isolated in the times D0, D30 and D60.

Discussion
Nowadays, anthropogenic activities may be one of the biggest reasons behind the dissemination of carbapenem resistance [15]. In this study, we describe the presence of KPC-producing Enterobacterales, in water, over time in Douro River, Vila Nova de Gaia, for 2 months.
CPE are more confined to healthcare settings, including occurrences of outbreaks, however, reports of these Enterobacterales have been increasingly reported in the environment [16]. All of the 15 isolates detected were classified as MDR, showing resistance to three or more classes of antibiotics in addition to meropenem resistance, which highlights an alarming resistance of these Enterobacterales strains. These MDR Enterobacterales are a public health concern due to their therapeutic importance, as the antibiotics to which they are resistant are some of the main therapeutic choices in the hospitals, particularly carbapenem resistant, which is mostly by transference of bla KPC [17,18]. These isolates were identified as 6 E. coli and 9 K. pneumoniae, all of them carrying bla KPC . As expected, bla KPC prevalence was higher in K. pneumoniae than E. coli. Data from the literature reports that Klebsiella spp. and E. coli are the most common carbapanemase producing Enterobacterales isolated from rivers and bla KPC the most prevalent in Europe [3,19], which is in line with our results.
K. pneumoniae is one of the pathogens that were given the highest "priority status", due to its potential drug resistance, including carbapenems [19]. In Portugal, an increasing trend of carbapenem resistance in K. pneumoniae has been described, evolving from 5.2% in 2016 to 11.6% in 2020, as reported by the European Centre for Disease Prevention and Control (ECDC) [20]. KPC-producing K. pneumoniae were the most prevalent in our study (9/15). Isolates harbouring these gene seem to be widespread in Portuguese hospitals [6,7]. Thus, the presence of bla KPC -harbouring K. pneumoniae in this study is in line with the epidemiology reported in Portuguese healthcare settings, which suggests an anthropogenic origin as the source of contamination.
E. coli are predominantly harmless bacteria that colonize the normal gut flora of humans, yet they are genetically diverse bacteria than can lead to extraintestinal infections [21,22]. Thus, the presence of E. coli resistant to carbapenems may severely affect human health, since it is known its capacity to carry several virulence factors and genes that are not present in strains belonging to commensal strains phylogroups [22]. The occurrence of E. coli KPC-producing strains in the aquatic environment is a worry, being distributed worldwide, although it is scarce, and it is described in countries with high rates of this carbapenemase in K. pneumoniae [18]. The emergence of bla KPC in E. coli has been linked to many mechanisms, such as plasmid exchange with other Enterobacterales, the global spread of genetically related strains and transpositional events in the species [18]. Therefore, the spread of E. coli carbapenem-resistant has severe implications in infection control, once compared with other CPE, since E. coli is a common bacteria in the community and healthcare settings and is easily transmitted.
KPC-producing Enterobacterales have been recurrently reported in hospitals sewage [5,16] and areas where industry is developed, such as agroindustry [3]. However, in our study, in a location as our sampling site, which is an important touristic spot where none of the above activities are practiced near, it is unusual and worrisome to find these reports. Fifteen KPCproducing Enterobacterales isolates were detected in this work with no clonal relationship, away from healthcare settings. The results suggest that the persistence of KPC-producing Enterobacterales was not related to clonal persistence. PFGE analysis showed the absence of a clonal relationship in the 15 isolates, which leads us to believe in the existence of a mobile genetic element responsible for the transmission of bla KPC between the different isolates. KPC-producing bacteria are one of the biggest challenges in healthcare settings worldwide [23]. The description of this public health threat in urban river waters, suggests environmental contamination by accidental healthcare setting sewage, once KPC-producing bacteria are characteristic of healthcare associated intestinal colonization. Our results suggest a punctual contamination with KPC-producing Enterobacterales continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. Gut colonization of the community population does not show this kind of threats and contamination of other zones of the river show contamination with coliforms but without expression of carbapenemases [24][25][26][27].

Sampling and Bacterial Isolation
Chronologically separated by 15 days (D0, D15, D30, D45, D60), five samples of water of Douro River were collected in the same place in Vila Nova de Gaia in 2021, from January to March.
The samples were stored directly in sterile glass bottles, held in the refrigerator and analysed shortly after collection (1 to 3 h).
We filtered 100 mL of the water samples, in duplicate, through sterile cellulose acetate membranes with 0.2 µm pore size (Frisenette, Knebel, Denmark) and placed them in MacConkey Agar (Liofilchem, Roseto degli Abruzzi, Italy) supplemented with meropenem (MRP, 0.5 µg/mL, Sigma-Aldrich, Darmstadt, Germany) to select carbapenem-resistant isolates [28]. Thereafter, the plates were incubated overnight at 37 • C. MacConkey Agar (Liofilchem, Roseto degli Abruzzi, Italy) without antibiotic, was used as growth control and control of the culture media was accomplished using Escherichia coli ATCCC 25922, a control strain.

Bacterial Identification and Antimicrobial Susceptibility Testing
One colony per morphology was picked from selective media onto the antibioticselection plate and incubated at 37 • C for 24 h.
The antimicrobial resistance profiles of the isolated bacteria were evaluated with 17 antibiotics by the agar diffusion method, according to Clinical and Laboratory Standards Institute (CLSI) [29]. Using antibiotic disks (Oxoid, Basingstoke, UK and Liofilchem, Roseto Phenotypic carbapenemase activity was evaluated by carbapenem inactivation method (CIM) which was performed on isolates showing reduced susceptibility to carbapenems. For a quick presumptive identification, CHROMagar Orientation (CHOROMagar, Paris, France) was performed and this identification was further confirmed with API 20E (Biomérieux, Marcy l'Etoile, France).

Beta-Lactamase Genes Screening
The bacterial DNA of carbapenem-resistant isolates was screened for carbapenemase genes. PCRs multiplexes were performed, which include the most prevailing acquired carbapenemase genes (bla KPC , bla OXA-48 , bla NDM , bla VIM and bla IMP ). The PCR was executed under the followed conditions: 10 min at 94 • C, followed of 36 cycles of 30 s at 94 • C, 40 s at 52 • C and 50 s at 72 • C and a final extension of 5 min at 72 • C [30], using Super Hot Master Mix (Bioron, Römerberg, Germany).
Additionally, PCR multiplex was also performed for the most prevailing ESBL genes, bla CTX-M group 1 , bla CTX-M group 2 , bla CTX-M group 8 , bla CTX-M group 9 , bla CTX-M group 25 , bla TEM , bla OXA and bla SHV . For bla CTX-M , the amplification conditions were initial denaturation at 94 • C for 5 min, 30 cycles of 94 • C for 25 s, 52 • C for 40 s and 72 • C for 50 s and 6 min at 72 • C [31]. Regarding bla TEM , bla OXA and bla SHV , the amplification was carried out at 94 • C for 10 min, 30 cycles of 94 • C for 40 s, 60 • C for 40 s, 72 • C for 1 min and a final elongation at 72 • C for 7 min [32].

Pulsed-Field Gel Electrophoresis
For clonality evaluation, PFGE was performed according to PulseNet USA standard protocol, through a CHIF-DR III system (BIO-RAD-U.S.A., Hercules, CA, USA) using 10 s as initial time and 40 s as final time for 21 h of running, after digestion of immobilized DNA with XbaI restriction enzyme (Bioron, Römerberg, Germany) [33].

Conclusions
In conclusion, our findings highlight the importance of AMR research in environmental samples and further analysis should be made to detect the possible causes of the presence and persistence of CPE in single specific location of this urban river. These results suggest a focal contamination, since it prevailed in the five samples, collected chronologically and separated by 15 days.
This work shows the relevance of this finding as a powerful biological marker of focal contamination and their persistence in river environments, by putative healthcare setting wastewaters, which may be related to the location and circulation of these genes by mobile genetic elements. These results alert to the need to expand the monitorization of carbapenemases dispersion in bacteria, particularly in natural environments exposed to anthropogenic pressures.