ESBL- and Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae among Bivalves from Portuguese Shellfish Production Areas

Bivalves are filter-feeding organisms and biomarkers of bacterial pollution. Our study aimed to analyze the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Escherichia coli among bivalves. A total of 522 bivalve samples were collected along Portuguese shellfish production areas. Homogenized samples were screened for E. coli contamination on corresponding selective plates, allowing for concomitant growth of Klebsiella pneumoniae. E. coli growth was observed in 39% of the samples. Subsequent selective screening identified nine samples (4.4%) contaminated with ESBL producers, corresponding to E. coli (n = 7) and K. pneumoniae (n = 2), while a single carbapenemase-producing K. pneumoniae (0.5%) was identified. ESBLs were all CTX-M-types commonly identified in human isolates, i.e., CTX-M-32 (n = 4), CTX-M-15 (n = 4), and CTX-M-14 (n = 1). The carbapenemase producer harbored the blaGES-5 gene located on a ColE plasmid. Clonality was evaluated by multilocus sequence typing, identifying E. coli backgrounds as ST10, ST23, ST540, ST617, ST746, SLV206, and SLV2325, commonly identified among environmental and human strains. The K. pneumoniae isolates belonged to ST834, ST15, and DLV644. The occurrence of ESBL- and carbapenemase-producing Enterobacteriaceae in bivalves reveals how the marine environment constitutes a reservoir of critical bacterial pathogens, thus potentially representing a risk to human health.


Introduction
The emergence of extended-spectrum β-lactamase (ESBL)-and carbapenemaseproducing Enterobacterales, and their subsequent spread, remains a major global threat. The transmission of such multidrug-resistant bacteria may occur not only from human to human, but also from animal sources to humans via the food chain [1]. In fact, high rates of Enterobacterales producing ESBLs or carbapenemases are seen frequently in foodproducing animals, namely in pigs, cattle, and poultry [2]. Although studies reporting ESBL-and carbapenemase-producing Enterobacterales in seafood products are scarce, more frequent surveillance studies have recently been initiated [3][4][5][6][7][8][9][10][11].
Marine bivalves represent a significant proportion of the world's seafood production sector with a global volume exceeding 15 million tons per year [12]. Due to its privileged geographical position, Portugal combines several characteristics favorable to the exploitation of bivalves: (1) 1793 km of coast under the influence of different currents; (2) a Mediterranean climate in the south and a temperate climate in the north; (3) average water temperatures ranging between 13 and 18 • C; and (4) the existence of several estuaries and lagoons along the coast [13]. The exploitation of bivalves arises from the north to the south of Portugal, with a significant impact on the national economy.
Bivalve species can be distributed in four major groups: clams, oysters, mussels, and cockles. These organisms feed by filtering phytoplankton and organic matter, a process that allows for the accumulation of various contaminants in these species, including bacteria of both aquatic and anthropogenic origin [14]. Bivalves are therefore good indicators of bacterial, as well as chemical, contamination in a given marine environment. In addition, since bivalves are often eaten raw or lightly cooked, they might constitute a risk for human health.
Bivalve production areas in Europe are assigned to different classes (A, B, or C) according to the content of fecal contamination. Class A areas meet the health standards, whereas class B and C areas require treatment to reduce microbiological contamination before marketing [15]. In Portugal, compliance in bivalve production areas is monitored weekly by the Instituto Português do Mar e Atmosfera (IPMA) and includes the screening for Escherichia coli contamination in bivalves collected in all production areas.
According to the European Centre for Disease Prevention and Control data of 2020, multidrug resistance among Enterobacterales remains a major problem in Portuguese hospitals [16]: (a) the mean proportion of ESBL-producing E. coli isolates causing human invasive infections was estimated at 14.4%; (b) the prevalence of ESBL producers in invasive Klebsiella pneumoniae was estimated at 47.6%; and (c) there has been a notorious, increasing trend in carbapenem resistance among K. pneumoniae, reaching 11.6% in 2020. Given this context, it is essential to better understand the sources and modes of dissemination of multidrug-resistant bacteria, especially those involved in human infections, using a global One Health approach, as recommended by the World Health Organization [17]. Following this approach, we have performed surveillance studies on antimicrobial resistance among multidrug-resistant Enterobacterales in different settings, i.e., among hospitalized patients and individuals at the time of hospital admission [18,19], among healthy community individuals (students) [20], among pigs [21], and among wild birds [22]. A single study conducted in 2019 evaluated the bacterial contamination in bivalves collected in Portuguese shellfish farms but did not report any ESBL producers among Enterobacteriaceae [23]. Therefore, the aim of the present study was to analyze the occurrence and characteristics of ESBL-and carbapenemase-producing E. coli and K. pneumoniae among bivalve samples collected in production areas from Portuguese estuaries and coastal waters, using the same prospective methodology as in our previous epidemiological studies.

Bivalve Samples
As part of the national monitoring program for shellfish production area management, 300 batch samples of bivalve mollusks were collected between December 2021 and September 2022 by IPMA from 19 different shellfish production areas along the Portuguese coast (12 estuaries and 7 coastal waters)- Figure 1 and Table 1. A total of 522 bivalve samples were collected.
Each batch sample comprised 10-20 individual bivalve specimens. Approximately 25 g of muscle and intervalvular liquid per bivalve sample were extracted into a sterile container using a scalpel, diluted in maximum recovery diluent (Oxoid, Heysham, Ireland), and homogenized for 60 s using a Stomacher 400 (Seward Laboratory System, London, UK). Homogenized samples were subsequently screened for E. coli contamination by plating onto Tryptone Bile X-glucuronide (TBX) selective plates (Oxoid), allowing for concomitant growth of K. pneumoniae. A total of 206 TBX plates showing presumptive E. coli growth were provided by IPMA for this study.

Bacterial Isolates
The bacterial growth of each TBX plate was resuspended in 3 mL of tryptic soy broth (TSB) (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) for enrichment and incubated at 37 • C overnight. Afterwards, a volume of 25 µL of each culture was inoculated into two selective media: (i) CHROMagar ESBL (Frilabo, Maia, Portugal) for ESBL producers, and (ii) ChromID Carba Smart selective medium (bioMérieux, La Balme-les-Grottes, France) for carbapenem-resistant isolates. For quality control, the selective agar plates were also inoculated with the following control strains: ESBL-positive K. pneumoniae ATCC 700603, carbapenemase-positive K. pneumoniae ATCC BAA-1705, and ESBL-and carbapenemasenegative E. coli ATCC 25922.
The isolates selected for the different media were identified at the species level using the API20E system (bioMérieux).

Molecular Analysis
Identification of ESBL and carbapenemase genes was performed by PCR as previously reported [26,27]. All positive amplicons were sent out for sequencing (Eurofins Genomics, Ebersberg, Germany).

Conjugation Experiments and Plasmid Analysis
Mating-out assays were performed using the azide-resistant E. coli J53 as the recipient. E. coli J53 and the bla GES-5 -positive donor were separately inoculated into 5 mL of tryptic soy broth (TSB (Frilabo)) and incubated at 37 • C for 5 h. The samples were subsequently mixed at a ratio of 1:4 (200 µL donor:800 µL recipient), centrifuged for 1 min at 3500 rpm, and 800 µL of the supernatant were discarded. The pellet was resuspended in the remaining 200 µL. This volume was deposited onto 22 µm filters onto a tryptic soy agar (TSA (Frilabo)) plate and incubated for 3 h. After 3 h, the filter was resuspended in 5 mL of 0.9% NaCl, and 100 µL of the mixture were plated onto TSA agar plates supplemented with cefoxitin (50 µg/mL) and azide (100 µg/mL). Susceptibility testing was performed for the E. coli transconjugants, and positivity for bla GES-5 was assessed by PCR.
The plasmid harboring the GES-5 carbapenemase-encoding gene was classified according to its incompatibility group using the PCR-based replicon typing (PBRT) method performed on DNA recovered from E. coli transconjugants, as described previously [29].

Statistical Analysis
Statistical analysis was performed using GNU R v 4.0.3 under RStudio 2022.07.1 + 554. Fisher's independence test was used to identify variables associated with the E. coli contamination and carriage of ESBL and carbapenemase producers. All p-values below 0.05 were considered statistically significant.

Population Description
Out of the 522 bivalve samples collected during a nine-month period, 206 (39%) grew E. coli on the TBX selective plates. Those 206 bivalve samples were recovered from the 19 production areas included in the study (Table 1). Of note, E. coli contamination rates were over 40% in 11 of the 19 production areas, all but one (L5b) being located in estuaries. In contrast, the production areas with lower contamination rates (<15%) were all situated in coastal waters (p < 0.001). However, the two coastal production areas closest to the Lisbon metropolitan area (L5a/b and L6) showed a significant amount (30-42%) of E. coli contamination.

Carriage of Multidrug-Resistant Enterobacterales
Out of the 206 samples recovered on the TBX plates, nine enterobacterial isolates grew on the CHROMagar ESBL agar, and one isolate grew on the ChromID Carba Smart selective medium.
The ESBL-producing isolates were recovered from six production areas (Table 1), out of which two showed higher frequencies of ESBL producers (p < 0.01 in both cases): ELM (22%) and ESD2 (17%)- Figure 1. The single carbapenemase-producing isolate was found in EMN. All ESBL-and carbapenemase-producing isolates were harvested from estuaries, although this result was not statistically significant (p = 0.16).

Discussion
Here we present a comprehensive study on the occurrence and characteristics of ESBLand carbapenemase-producing isolates among bivalve samples, during a nine-month period, from different shellfish production areas in Portuguese estuaries and coastal waters. Our study revealed that a high proportion (39%) of bivalves were contaminated with E. coli, and among these, 4% were contaminated with ESBL producers. In addition, one sample was contaminated with a carbapenemase-producing K. pneumoniae. Of note, all ESBL-and carbapenemase-producing isolates were harvested from estuaries, where effluent flow is less affected by tides and there is lower salinity compared to coastal waters.
The rates of ESBL-producing isolates in the present study were slightly higher than those reported among bivalves in other countries: (i) a study on antibiotic resistance of E. coli from marine bivalves collected in Norway identified ESBL-encoding genes in 1% of isolates (2/199) [5], and (ii) 1.6% of clams bought from retail markets in Tunisia were contaminated with ESBL-producing Enterobacterales [9]. In contrast, the proportion was much lower than the 15% (21/141) reported among E. coli isolates recovered from clams in Italy [6] and India, where 53% of fresh shellfish from retail markets exhibited ESBL-producing E. coli isolates [11].
All ESBLs found in the present study belonged exclusively to the CTX-M family, i.e., CTX-M-32, CTX-M-15, and CTX-M-14. These enzymes are commonly identified in human isolates worldwide, including Portugal [19,20]. Accordingly, we may speculate that the ESBL producers identified among bivalves might correspond to strains colonizing the human gut.
One out of the three K. pneumoniae identified in the present study displayed a carbapenemase gene, which is usually much less frequent among bivalve samples than are ESBLs. Sporadic carbapenem-producing Enterobacterial isolates were previously reported: In Germany, one VIM-1-producing E. coli was isolated from clams collected in Italy [10]. In Tunisia, two K. pneumoniae isolates (bla NDM-1 and bla OXA-48 ) were isolated in clams, and a KPC-3-producing E. coli was recovered in mussels purchased in retail markets [8,9]. In Myanmar, two NDM-1 producers (one E. coli and one K. pneumoniae) were isolated from a clam and a prawn, respectively [30], and in Canada, three and two clam samples imported from Vietnam contained E. cloacae harboring bla IMI-1 and bla NDM-1 , respectively [31]. We identified in the present study the first enterobacterial isolate producing a carbapenemase of GES-type (GES-5) recovered from bivalve samples. This carbapenemase has been previously detected in Portugal among humans [18], gulls [22], the aquatic environment [32], and now bivalves.
The seven ESBL-producing E. coli strains belonged to distinct genetic backgrounds (seven STs), which suggests multiple sources of E. coli contamination in the harvesting production areas. Most of these STs have been previously found among humans and/or animals and/or in the environment, namely in Portugal. In fact, ST10 was the predominant clone among E. coli recovered from healthy students in Lisbon [20], was frequently isolated in Portuguese hospitals [33], and was found among healthy and sick cats in the country [34]. Moreover, ST10 was the predominant lineage among E. coli present in irrigation water and vegetables from 16 household farms in Portugal [35]. Concerning bivalves, one E. coli ST10 producing VIM-1 was isolated from a clam harvested in the Mediterranean Sea (Italy) [10]. ST617, which also belongs to CC10, has been formerly reported in Portugal in a CTX-M-55 E. coli strain colonizing a healthy dog [36] and from two CTX-M-15 E. coli strains isolated from the effluent from a wastewater treatment plant [37]. In addition, ST617 has been previously described in a CTX-M-14 E. coli isolate from clams sold in Tunisian markets [9].
In the present study, one E. coli isolate belonged to a single-locus variant (SLV) of ST206. This ST has been reported only once in Portugal, corresponding to a single CTX-M-15 isolate recently recovered from a pigeon fecal sample in Lisbon [38]. ST206 is mainly associated with animals, being the main clone among chickens in Nigeria [39] and frequently found among food-chain animals in China [40,41]. Concerning E. coli ST23, it has also been previously reported in Portugal among hospital outpatients [33]. This ST was frequently found among human isolates, namely causing urinary tract infections (UTIs) in nursing homes in the Netherlands [42] and also causing mastitis in cattle in France [43].
One ESBL-producing K. pneumoniae strain found in the present study belonged to ST15. ST15 was the main (32%) clonal type among 509 K. pneumoniae isolates collected nationwide in Portugal from 16 hospitals and environmental settings between 1980 and 2019 [44]. ST15 includes clinical, animal, and environment isolates, being an international lineage with a wide geographic distribution [45]. Overall, the clinically relevant bacteria identified in our study seem to qualitatively mirror the human panorama, not only in terms of β-lactamases circulating in this specific environment, but also in terms of clonal backgrounds. These results suggest a very likely human fecal contamination, possibly through sewage effluents that are discharged in estuaries where bivalve production areas are located.
In conclusion, this study constitutes the first report of enterobacterial ESBL and carbapenemase producers among bivalves in Portuguese coastal waters. Although a relatively low occurrence of ESBL and carbapenemase producers was detected, our study demonstrates that bivalves collected for human consumption may act as a potential reservoir of multidrug-resistant bacterial pathogens that eventually may be transmitted through the food chain. Therefore, in the future, the compliance of bivalve production areas routinely monitored by IPMA should also include screening for ESBL-and carbapenemase-producing Enterobacterales contamination in bivalves collected in the different production areas. Funding: This work was partially financed by the Portuguese Red Cross, by the University of Fribourg, and by the Swiss National Science Foundation (projects FNS-31003A_163432 and FNS-407240_177381).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are all available in the main text.

Conflicts of Interest:
The authors declare no conflict of interest.