# Do Microorganisms in Bathing Water in Guadeloupe (French West Indies) Have Resistance Genes?

Degrâce Batantou Mabandza, Edlyne Colletin, Christophe Dagot, Isaure Quétel, Sébastien Breurec, Stéphanie Guyomard-Rabenirina

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Degrâce Batantou Mabandza, Edlyne Colletin, Christophe Dagot, Isaure Quétel, Sébastien Breurec, et al.. Do Microorganisms in Bathing Water in Guadeloupe (French West Indies) Have Resistance Genes?. Antibiotics, 2024, 13 (1), pp.87. 10.3390/antibiotics13010087. hal-04666344

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Article

# Do Microorganisms in Bathing Water in Guadeloupe (French West Indies) Have Resistance Genes?

Degrâce Batantou Mabandza <fnref n="1" />,*, Edlyne Colletin <fnref n="1" />, Christophe Dagot <fnref n="2" />, Isaure Quétel <fnref n="1" />, Sébastien Breurec <fnref n="1" />,<fnref n="3" />,<fnref n="4" />,<fnref n="5" />,<fnref n="6" />
and Stéphanie Guyomard-Rabenirina <fnref n="1" />

**Abstract:** Waterborne faecal contamination is a major public health concern. The main objectives of this study were to investigate faecal contamination and *Escherichia coli* (*E. coli*) antibiotic resistance in recreational fresh water from Guadeloupe and to characterise the microbiome and resistome composition in biofilms from submerged rocks. Significant faecal contamination was observed at 14 freshwater sites. *E. coli* predominated (62%), followed by *Enterobacter cloacae* (11%) and *Acinetobacter* spp. (11%). Of 152 *E. coli* isolated, none produced extended-spectrum beta-lactamases (ESBLs), but 7% showed resistance to streptomycin and 4% to tetracycline. Biofilm resistome analysis revealed clinically significant antibiotic-resistance genes (ARGs), including those coding for resistance to sulfonamides (*sul1*), carbapenems (*bla*<sub>KPC</sub>), and third-generation cephalosporins (*bla*<sub>CTX-M</sub>). Mobile genetic elements (MGEs) (*intI1*, *intI2*, *intI3*) linked to resistance to aminoglycosides, beta-lactams, tetracycline, as well as heavy metal resistance determinants (*copA*, *cusF*, *czcA*, *merA*) conferring resistance to copper, silver, cadmium, and mercury were also detected. Diverse bacterial phyla were found in biofilm samples, of which Proteobacteria, Bacteroidetes, Planctomycetes, and Cyanobacteria were predominant. Despite the frequent presence of *E. coli* exceeding regulatory standards, the low levels of antibiotic-resistant bacteria in freshwater and of ARGs and MGEs in associated biofilms suggest limited antibiotic resistance in Guadeloupean recreational waters.

**Keywords:** *Escherichia coli*; resistome; biofilm; river; pollution

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**Citation:** Batantou Mabandza, D.; Colletin, E.; Dagot, C.; Quétel, I.; Breurec, S.; Guyomard-Rabenirina, S. *Do Microorganisms in Bathing Water in Guadeloupe (French West Indies) Have Resistance Genes?* *Antibiotics* **2024**, *13*, 87. https://doi.org/10.3390/antibiotics13010087

Academic Editor: Juhee Ahn

Received: 14 December 2023

Revised: 5 January 2024

Accepted: 11 January 2024

Published: 16 January 2024

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**Copyright:** © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Antibiotics are commonly used in human and veterinary medicine to treat or prevent bacterial infections [1,2]; however, the over- and inappropriate use of these drugs has led to a rise in antibiotic resistance in bacteria. Non-antibiotic compounds, such as antibacterial biocides and heavy metals, can also contribute to the rise in antibiotic resistance through co-selection mechanisms [3]. Horizontal gene transfer (HGT), facilitated by mobile genetic elements (MGEs) like integrons, transposons, and plasmids, enables the exchange of resistance-conferring genes to these elements [4]. Antimicrobial resistance (AMR) is now a major public health problem worldwide [5–7].

Guadeloupe, a French overseas tropical island in the Caribbean, is a major element in human migration between Europe, the USA, and other Caribbean islands [8]. With its high economic level (https://hdr.undp.org, accessed on 9 March 2023), insularity, small surface area (1436 km<fnref n="2" />), and small population (375,845 inhabitants in 2023), it is a propitious

<footnote n="1">¹ Transmission, Reservoir and Diversity of Pathogens Unit, Pasteur Institute of Guadeloupe, 97110 Pointe-à-Pitre, France</footnote>

<footnote n="2">² University of Limoges, INSERM, CHU Limoges, RESINFIT, U1092, 87000 Limoges, France</footnote>

<footnote n="3">³ Faculty of Medicine Hyacinthe Bastaraud, University of the Antilles, 97110 Pointe-à-Pitre, France</footnote>

<footnote n="4">⁴ INSERM, Centre for Clinical Investigation 1424, 97110 Pointe-à-Pitre, France</footnote>

<footnote n="5">⁵ Department of Pathogenesis and Control of Chronic and Emerging Infections, University of Montpellier, INSERM, 34394 Montpellier, France</footnote>

<footnote n="6">⁶ Laboratory of Clinical Microbiology, University Hospital Centre of Guadeloupe, 971110 Pointe-à-Pitre, France</footnote>

<footnote>* Correspondence: dbatantou@pasteur-guadeloupe.fr</footnote>
environment for studying reservoirs of antibiotic-resistant isolates and identifying routes
of transmission to the human population.

Data on resistance to antibiotics in Guadeloupe are scarce and recent. The island has faced the emergence of nosocomial infections associated with carbapenemase-producing Enterobacteriaceae since 2014 and also a high incidence of nosocomial infections caused by ESBL-producing Enterobacteriaceae [9–12]. Environmental investigations have focused on the faecal carriage of ESBL-producing Enterobacteriaceae in domestic animals [13,14], farm animals [15], and wildlife [16,17] and also on the transmission of clinical strains to the environment [8,18]. Antibiotic resistance was also studied in effluents from wastewater treatment plants (WWTP) and in surface waters with or without such discharges [18,19]. Although these studies concluded that there is limited circulation of antibiotic-resistant bacteria (ARB) between humans and the environment [8,13,16,18], there are still gaps in understanding the transmission of ARB from the environment to humans.

Guadeloupe, renowned for its beaches and rivers, welcomes large numbers of tourists, with over 815,000 visitors in 2019 [20], and aquatic recreation plays a central role in tourism. Monitoring water quality is therefore essential. The Regional Health Agency has mandated the Pasteur Institute of Guadeloupe to conduct monthly monitoring of the bacteriological quality of all the beaches and rivers of the archipelago on which aquatic recreation is practised. Microbiological water quality control is based on the monitoring of faecal contamination indicators such as *E. coli* and intestinal enterococci [21]. In 2020, 11% of Guadeloupe's recreational waters did not meet the required quality standards (59% of river monitoring sites), and bathing was prohibited at 3% of the sites [22]. Despite growing concern about faecal pollution of water in Guadeloupe, data on AMR in recreational areas that do not receive WWTP discharges are scarce. The only study, conducted in 2016 [19], revealed limited antibiotic resistance in such water; however, the conclusion was limited to surface waters and did not address the issue of biofilms.

It is now well established that ARB and their resistance determinants can infiltrate surface waters [23–26] and biofilms [27–29]. Biofilms can adhere to a variety of surfaces, including rocks in rivers. Such bacterial communities form a complex extracellular matrix that confers high resistance to antimicrobial treatment. Previous studies have reported the presence of ARB in river biofilms, sediments, and the water column along river systems [30–32].

The aim of this exploratory study was to assess the contamination of recreational river surface water and biofilms by antibiotic-resistant coliforms and broaden the understanding of antibiotic resistance on the island of Guadeloupe. The main objective was to characterise antibiotic resistance in 14 rivers known to have poor water quality. In addition, we analysed the microbiome and resistome of biofilms present on rocks at the bottom of three of the most polluted rivers. Surface water contamination by faecal coliforms was estimated by culturing *E. coli*, and the characterisation of phenotypic resistance was limited to this species. Unlike enterococci, antibiotic-resistant *E. coli* can be considered the sentinels of AMR in the environment [33,34]. The abundance of resistance determinants was assessed by Fluidigm polymerase chain reaction (PCR) in biofilms, and the microbiome was characterised by metabarcoding targeting the 16S rRNA gene.

**2. Results**

2.1. *Enumeration of E. coli in Water Samples*

*E. coli* were counted by the "most probable number" (MPN) method. Of 59 river sam-
ples, 48 had an MPN of <100 colony-forming units (CFUs)/100 mL, of which 15 had none.
The lowest value in 11 samples with MPN values > 100 CFU/100 mL was 110 CFU/100 mL
(Grande Rivière du Lamentin) and the highest value was 2508 CFU/100 mL (Corossol).
Only six sites had high levels of faecal bacteria, and no *E. coli* were found at Sofaïa (Table 1).
During campaign 3, when there was heavy rainfall, *E. coli* levels were >1000 CFU/100 mL
at only three sites (Table 1).
<table><caption>**Table 1.** Numbers of *E. coli* (most probable number, CFU/100 mL) in river water in Guadeloupe.</caption><thead><tr><th rowspan="2">Municipality</th><th rowspan="2">Site</th><th colspan="5">Most Probable Number (CFU/100 mL)</th></tr><tr><th>Campaign 1</th><th>Campaign 2</th><th>Campaign 3</th><th>Campaign 4</th><th>Campaign 5</th></tr></thead><tbody><tr><td>Deshaies</td><td>Fond Helliot</td><td>30</td><td>49</td><td>144</td><td>77</td><td>347</td></tr><tr><td>Gourbeyre</td><td>Bain des Amours</td><td>0</td><td>15</td><td>61</td><td>61</td><td>30</td></tr><tr><td>Gourbeyre</td><td>Bassin Bleu</td><td>-</td><td>75</td><td>61</td><td>15</td><td>46</td></tr><tr><td>Gourbeyre</td><td>Bain de Dole</td><td>46</td><td>0</td><td>46</td><td>30</td><td>160</td></tr><tr><td>Lamentin</td><td>Grande Rivière du Lamentin</td><td>110</td><td>2213</td><td>179</td><td>144</td><td>127</td></tr><tr><td>Petit Bourg</td><td>Bras David</td><td>15</td><td>15</td><td>1336</td><td>15</td><td>-</td></tr><tr><td>Petit Bourg</td><td>Cascade aux Ecrevisses</td><td>15</td><td>15</td><td>30</td><td>46</td><td>61</td></tr><tr><td>Petit Bourg</td><td>Corossol</td><td>0</td><td>-</td><td>2508</td><td>0</td><td>46</td></tr><tr><td>Petit Bourg</td><td>Diane</td><td>15</td><td>46</td><td>77</td><td>77</td><td>-</td></tr><tr><td>Petit Bourg</td><td>Duquerry</td><td>0</td><td>15</td><td>15</td><td>15</td><td>-</td></tr><tr><td>St Claude</td><td>Bain Jaune</td><td>-</td><td>-</td><td>30</td><td>0</td><td>0</td></tr><tr><td>St Claude</td><td>Rivière Rouge</td><td>-</td><td>15</td><td>94</td><td>0</td><td>0</td></tr><tr><td>St Rose</td><td>Sofaïa</td><td>-</td><td>0</td><td>-</td><td>0</td><td>0</td></tr><tr><td>Vieux Habitants</td><td>Vallée Verte</td><td>-</td><td>0</td><td>1071</td><td>0</td><td>0</td></tr></tbody></table>

- Not collected.

## 2.2. Characterisation of Bacterial Isolates from River Water, Antibiotic Susceptibility of E. coli, and Resistance in Biofilms

Blue colonies were confirmed as *E. coli* on chromogenic media by matrix-assisted laser desorption/ionisation mass spectrometry. Other colonies of various colours were selected randomly for the identification of bacterial diversity in bathing water. During the five sampling campaigns, 245 bacteria were identified, the most common species being *E. coli* (*n* = 152, 62%), followed by *Enterobacter cloacae* (*n* = 25, 11%) and *Acinetobacter* spp. (*n* = 25, 11%). Other relevant species (Citrobacter freundii, C. amalonaticus, Klebsiella pneumoniae, K. variicola, K. oxytoca, K. ascorbata, Aeromonas hydrophila, Ae. enteropelogenes, Ae. jandaei Pseudomonas putida, Serratia marcescens, Chromobacterium violaceum, Pseudomonas alcalifaciens, Providencia alcalifaciens, Raoultella ornithinolytica, and Cronobacter spp.) were less abundant.

No *E. coli* strains were detected in the medium with added third-generation cephalosporin. None of the 47 *E. coli* strains tested for antibiotic susceptibility showed resistance, except for streptomycin (*n* = 3, 7% resistance) and tetracycline (*n* = 2, 4% resistance). These results suggest little bacterial resistance in recreational river waters from Guadeloupe. In order to confirm these results, we characterised the resistome of biofilms present in the three most polluted rivers (Corossol, Fond Helliot, and Grande Rivière du Lamentin).

In two campaigns, nine categories of clinical resistance genes associated with streptogramin, macrolide, sulfonamide, polymyxin, tetracycline, beta-lactam, aminoglycoside, quaternary ammonium compounds (QAC), and heavy metal resistance were identified. In addition, MGEs were present at all three sites (Figure 1a). The relative abundance of resistance determinants and MGEs differed in the two sampling campaigns, especially at the Corossol and Fond Helliot sites (Figure 1a). Sulphonamide resistance genes were more abundant during the first campaign (30 May 2022) (Figure 1a), while beta-lactam, aminoglycoside, heavy-metal resistance genes, and MGEs were more prevalent in the second campaign (5 July 2022) (Figure 1a). No major difference was observed between the two campaigns for the other resistance determinants, with the exception of tetracycline and QAC resistance genes, which were particularly abundant at the Fond Helliot site during the second campaign (Figure 1a).
<figure><img src="image_5.png" /><figcaption>**Figure 1.** (a) Normalised relative abundance of antimicrobial classes (antibiotic-resistance gene [ARG] copies per 16S rRNA gene copy) in six biofilm samples. (b) Heat map of the relative abundance of the 64 ARGs detected in the six biofilm samples (ARG copy per 16S rRNA gene copy). White bands represent undetected elements.</figcaption></figure>

A total of 64 subtypes of resistance determinants were identified in the samples analysed, although in small quantities (Figure 1b). The number of ARG subtypes varied
from 34 (minimum) to 49 (maximum) per sample (Figure 1b) and the abundance from
$3.30 \times 10^{-7}$ to $3.33 \times 10^{-3}$ copies/16S rRNA gene. Similar numbers of ARGs and MGE
were detected at all sites. The Fond Helliot site had a higher overall relative abundance
than the other sites on 30 May 2022 (Figure 1b). Overall, the signal intensity of resistance
determinants and MGEs was more pronounced during the first campaign (Figure 1b),
particularly at Grande Rivière du Lamentin and Fond Helliot, while in the second cam-
paign, signal intensity was higher at Corossol. The five most abundant subtypes at all
the sites in both campaigns were the resistance genes *sul1*, *aac(3)-Iid*, *bla*<sub>KPC</sub>, *bla*<sub>CTX-M</sub>, and *bla*<sub>VIM</sub> (Figure 1b), although the abundance of *sul1* was greatest at Fond Helliot in the
first campaign. The MGEs identified were inc-P1 plasmid, transposon Tn3 (*tnpA*), inser-
tion sequences (*IS*6 family, *ISS1*, *ISS1W*), and integrons including *intl1*, *intl2*, and *intl3*
(Figure 1b), some of which were ubiquitous, while others were detected only at certain
sites. Thus, the analysis of biofilm resistomes from the rivers studied indicated only limited
resistance determinants.

## 2.3. *Composition of Bacterial Communities in Biofilms*

After quality filtering, 1,039,932 normalised sequences were obtained from six biofilm
samples, i.e., 173,322 sequences per sample. An analysis of the 16S sequencing data in-
dicated the presence of several bacterial communities in river water biofilms. A total of
13,404 amplicon sequence variants was found in the prokaryotic communities. 16S rRNA
gene sequencing showed that these bacterial communities comprised 40 phyla, 99 classes,
223 orders, 320 families, and 740 genera. The bacterial communities in biofilms from the
Corossol, Fond Helliot, and Grande Rivière du Lamentin rivers were similar at the phylum
(Figure 2a) and genus (Figure 2b) levels, despite their geographical separation, although
the relative abundance varied. Proteobacteria (50% of all phyla) were the dominant phylum
in all samples (Figure 2a). The major phyla at other sampling sites or campaigns were Bac-
teroidetes (10% of all phyla), Planctomycetes (9% of all phyla), and Cyanobacteria (8% of all
phyla) (Figure 2a), and the predominant bacterial genera were *Rhizobiales*, *Burkholderiaceae*,
*Exiguobacterium*, and *Saprospiraceae*, accounting for 3.5–15.1% of all genera (Figure 2b).

Variations in taxonomic relative abundance were observed between the two sampling
campaigns. For example, the relative abundance of the Cyanobacteria and Deinococcus-
Thermus phyla decreased between the first (30 May 2022) and second (5 July 2022) campaign
(35% (sum of relative abundances) vs. 11%, 20% vs. 2%, respectively), while the relative
abundance of Firmicutes increased (3% vs. 35%), especially at the Fond Helliot and Grande
Rivière du Lamentin sites (Figure 2a). At the genus level, *Exiguobacterium* was more
abundant at both sites during the second sampling campaign (Figure 2b). *Truepera* and
*Deinococcaceae* were detected in low proportions during the first sampling campaign and
almost disappeared during the second campaign (Figure 2b). A similar trend was observed
for *Saprospiraceae* at the Corossol and Grande Rivière du Lamentin sites (Figure 2b). In
contrast, the relative abundance of *Rhodobacter* increased at all sites, from a total relative
abundance of 6% in the first campaign to 15% in the second (Figure 2b). Geographical
variations were also observed. The *Rhizobiales*, *Burkholderiaceae*, and *Acidimicrobiia* genera
were predominant at the Fond Helliot site (Figure 2b).

## 2.4. *Analysis of Alpha Diversity*

Analysis of variance of the Shannon index showed a significant difference in alpha
diversity among the sites (p = 0.03) but not among sampling dates (p = 0.88). In pairwise
comparisons (post hoc Tukey) of similarities, samples from Fond Helliot differed signifi-
cantly from those from Corossol (p = 0.02), while those from Fond Helliot-Grande Rivière
du Lamentin and Grande Rivière du Lamentin-Corossol did not (p = 0.15 and p = 0.14,
respectively). These results indicate variations in the specific richness and equitability of
communities among sites (Figure 3). The Shannon diversity index, calculated for each
sample, confirmed that the Corossol site had the highest bacterial diversity of the three
sites (Figure 3).
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Normalised relative abundance of bacteria at phylum (a) and genus (b) level in six biofilm samples from the Corossol, Fond Helliot, and Grande Rivière du Lamentin rivers on 30 May 2022 and 5 July 2022. NA means not assigned. For names with NA, the genera have not been identified. We have referred to the last taxonomic assignment found.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Analysis of alpha diversity. The graph shows the average richness and the Shannon index.</figcaption></figure>

## 2.5. *Analysis of Beta Diversity*

Beta diversity analysis (Figure 4) confirmed the similarity of bacterial composition among the sites. No significant differences in the structure of the bacterial community were observed at the different sampling sites ($p = 0.70$) or between the sampling dates ($p = 0.07$).

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Analysis of beta diversity with the Bray-Curtis index.</figcaption></figure>
### 3. Discussion

This study focused on the presence of total and resistant *E. coli* in recreational freshwater unaffected by effluent from wastewater treatment plants, with *E. coli* being found at least once at all study sites except one. Of the 14 sites, 11 had *E. coli* at levels >100 CFU/100 mL, indicating poor water quality and the presence of faecal pollution. During the third campaign, increased *E. coli* levels were found at Bras David, Corossol, and Vallée Verte, indicating that precipitation contributes to soil leaching, with discharge of bacteria into aquatic environments [35,36]. These high *E. coli* loads could be linked to discharges of wildlife faeces, particularly from birds. A low level of antibiotic resistance was found in the *E. coli* isolates studied: no ESBL production was observed and only resistance to streptomycin and tetracycline was detected, due probably to their extensive use in veterinary medicine [37,38]. The results are in line with previous local data indicating a low level of antibiotic resistance in areas with no anthropogenic impact [19]. In contrast, in the Netherlands, ESBL-producing *E. coli* were detected in surface waters not influenced by WWTPs in similar concentrations than those under the influence of WWTPs, indicating the existence of additional ESBL-producing *E. coli* contamination sources [39].

ARGs were detected in biofilms from all river sites, although at low levels, and were associated with resistance to several classes of antibiotics. Interestingly, 28 ARG subtypes were common to all samples, indicating a similar ARG composition. ARGs were associated with resistance to carbapenem and polymyxin, two clinically relevant last-line treatments for life-threatening infections [40,41]. Determinants of resistance to tetracyclines, sulfonamides, beta-lactams, aminoglycosides, and macrolides were also detected. Interestingly, *bla*<sub>CTX-M</sub> was one of the most frequent ARGs detected, although no bacteria resistant to third-generation cephalosporin were isolated.

The integrons *intl1*, *intl2*, and *intl3* detected here have previously been described as having a clinical origin [4,42,43], with *Intl1* potentially being used as an indicator of environmental resistance [44]. These integrons are mainly linked to horizontal gene transfer among several bacterial species [43]. *intl3* was the only integron identified consistently across all sites and during both sampling periods, and its presence was associated with the soil and freshwater proteobacterial group [42]. We also identified determinants of resistance to heavy metals and disinfectants, which can accelerate the spread of ARGs and antibiotic-resistant bacteria while promoting horizontal gene transfer [45-49]. Although the sample used in this study was relatively small for a resistome study, our results suggest that biofilms in these rivers are not sites of permanent ARG accumulation or establishment. More exhaustive sampling would provide a better understanding of environmental impacts such as the spatial and temporal extent of waterborne bacteria carrying ARGs. Nevertheless, our analysis suggests no permanent risk for human health from antibiotic resistance in Guadeloupe's recreational waters, including biofilms.

An analysis of the biofilm microbiome characteristics from three freshwater sites showed that the predominant bacterial phyla at our study sites were Proteobacteria, Bacteroidetes, Planctomycetes, and Cyanobacteria, as reported in previous studies but with different abundances [50–55]. The predominance of Proteobacteria was highlighted in previous studies investigating surface waters [56–61] and biofilms from various environments [51,62,63] through 16S rRNA gene analysis. With Proteobacteria considered prolific surface colonisers [64], previous studies have also revealed their predominance in aquatic ecosystems, where they influence biogeochemical cycles such as those of carbon and nitrogen [55,65–67]. This is in agreement with the large amounts of nutrients in our freshwater sites, which favour their proliferation. Bacteroidetes have been associated with biopolymer degradation and contribute to dissolving organic matter [68], while Planctomycetes have been associated with ammonium regulation [69]. Cyanobacteria, a group of aquatic and photosynthetic bacteria, influence the structure and productivity of microbial communities [70]. The *Rhizobiales* and *Burkholderiales* genera were the most abundant, indicating oligotrophic or mesotrophic conditions [71,72]. The genus *Saprospiraceae*, which was also abundant, is commonly associated with wastewater and sewage sludge [73,74]. It is also
found in various marine and freshwater environments [74], where it can hydrolyse and use complex organic carbon compounds [75]. Although the residence time of submerged rocks for river biofilm formation was the same in both sampling campaigns, variations in the relative abundances of phyla and genera were observed within the same site. The decrease or increase in intra-river relative abundance of specific taxa between the two sampling periods suggests a sensitivity to specific environmental parameters. Ecological interactions, nutrient availability, and local environmental conditions (T °C, pH, etc.) are factors likely to influence this dynamic [54,64,76]. Other studies have indicated that water flow [77] and oxygenation [78] contribute to the shaping of microbial communities in biofilms. Combined with temporal heterogeneity, geographic heterogeneity was observed with genera present in higher proportions at some sites than at others, in agreement with previous observations [79,80].

Statistical analysis of alpha diversity showed significant differences among sites (p = 0.03), indicating variations in species richness and equitability. No significant difference in diversity was observed between sampling dates (p = 0.88), suggesting that species diversity remained relatively stable during this time; however, a longer study period would be necessary to reach conclusions about the stability of the microbiota over time. Our results on diversity among the sites are in line with those of Zancarini et al. [81], who observed significant variations in the composition of the microbial community between collection points up and down a 122 km river. The variations were attributed to differences in water temperature, with an average difference of 5 °C between upstream and downstream sites, and also in the geological and hydrological characteristics of the sampling sites [81]. In our study, no significant difference was found in beta diversity between sites or sampling dates, although subtle variations in bacterial community structure were observed between sampling dates (p = 0.07). These observations suggest subtle changes in the composition of bacterial communities over time, which could be linked to environmental factors such as water temperature [82–84] or to events that influence the structure of microbial biofilms in freshwater samples. These results contrast with those of two previous studies, in which significant differences were observed between sampling sites [81,85] but not between sampling dates [81], which suggests that the composition of the microbial community is relatively stable over time but may vary according to the characteristics of each sampling site [81,84]. Other studies have also shown that the composition of phyla within biofilms can be influenced by pollution, seasonal variations and the physico-chemical properties of the water [54,64,76,86]. These observations highlight the sensitivity of biofilm communities not only to geographical location, and thus the environment, but also to time, particularly seasons [82,84,87].

# 4. Materials and Methods

## 4.1. *Sampling of River Water and Biofilms*

Five sampling campaigns to collect river water were carried out between March and June 2022, coinciding with the dry season. Fourteen rivers in seven municipalities were selected in 2021 on the basis of data from annual monitoring of bathing water quality by the Regional Health Agency for frequent poor water quality (100 > *E. coli* CFU/100 mL ≥ 1800). Water samples were taken approximately 30 cm below the surface in a sterile container, always at the same GPS point. All the rivers are located on the island of Basse-Terre and are in the same hydro-ecoregion, a homogeneous zone with respect to geology, climate, and landscape (Figure 5). Each site was sampled at least three times during the five campaigns.

Two 3-week campaigns to collect biofilms were conducted in May and June 2022. The three rivers selected because of their average or poor water quality were Grande Rivière du Lamentin (16.22603° N; 61.66128° W), Corossol River (16.17071° N; 61.68838° W), and Fond Helliot River (16.28408° N; 61.79241° W).

A total of fifty-nine river water samples and six biofilm samples were taken for bacteriological analysis. All samples were placed in a refrigerated container (4 °C) during
transport and protected from sunlight until their delivery to the laboratory. The samples
were then kept in a refrigerator at 4 °C and analysed within 24 h.

<figure><img src="image_10.png" /><figcaption>**Figure 5.** Map of Guadeloupe and location of the sampling sites in the seven municipalities (in green) in the southern part of the Island.</figcaption></figure>

4.2. *Enumeration of Escherichia coli in Water*

The MPN of *E. coli* was determined on MUG microplates (Bio-Rad, Marnes-la-Coquette, France) according to NF EN ISO 9308-3:1998 [88]. To a tube containing 18 mL of special microplate diluent, 18 mL of sample was added to obtain a 1:2 dilution. The tube was vortexed and 2 mL of the suspension was removed and added to another tube containing 18 mL of special microplate diluent to obtain a final dilution of 1:20. For enumeration of third-generation cephalosporin-resistant *E. coli*, 1 mL of ceftriaxone antibiotic solution was added to the dilutions for a final concentration of 4 mg/L.

For each river water sample, a microplate containing dehydrated *E. coli*-specific culture medium was halved. The 1:2 dilution was distributed into 64 wells and the 1:20 dilution into the remaining 32 wells with an eight-channel pipette set at 200 µL. Plates were covered with sterile adhesive strips and incubated at 44 °C for 36-72 h, and the number of positive wells was converted into the MPN from the MPN table. The values obtained were compared with the standard threshold values provided in the European Directive of 15 February 2006 (2006/7/EC) [89] and the Public Health Code: Articles L.1332-1 to L.1332-9 and D.1332-14 [90,91].

4.3. *Isolation, Identification, and Susceptibility of E. coli in Water*

*E. coli* strains were isolated by the membrane filtration method as described previ-
ously [19]. Filters and positive wells were inoculated onto chromogenic coliform agar
medium either without antibiotics or supplemented with ceftriaxone at 4 mg/L. After
incubation for 24 h at 37 °C, 10 dark blue CFUs (presumptive *E. coli* onto chromogenic
coliform agar) were plated on non-selective bromo-cresol purple agar. Blue presumed *E. coli*
colonies were identified by matrix-assisted laser desorption/ionisation mass spectrometry,
and colonies of other colours were selected at random for this exploratory research. After
species confirmation, a maximum of three *E. coli* colonies was collected from media on
which growth was observed, for a total of 152 *E. coli* isolates. One strain per sample (n = 47)
from the agars on which bacterial growth had been observed was chosen arbitrarily for anal-
ysis of antibiotic resistance phenotypes. The disc diffusion technique on Mueller–Hinton agar was used according to the recommendations of the Antibiogram Committee of the French Society of Microbiology [92] (http://www.sfm-microbiologie.org, accessed on 18 July 2023).

The strains were tested against a panel of 16 antimicrobial agents corresponding to those most commonly prescribed in human and veterinary medicine: amikacin (30 µg), ciprofloxacin (5 µg), amoxicillin/clavulanic acid (20 µg/10 µg), ampicillin (10 µg), cefoxitin (30 µg), ceftazidime (10 µg), cefotaxime (5 µg), ertapenem (10 µg), fosfomycin (200 µg), gentamicin (10 µg), nalidixic acid (30 µg), streptomycin (10 µg), temocillin (30 µg), tetracycline (30 µg), ticarcillin (75 µg), and trimethoprim/sulfamethoxazole (25 µg). Growth inhibition diameters were measured with the Adagio automated system (Bio-Rad). The critical diameter reference proposed by CA-SFM/EUCAST 2021 [92] was used to interpret the diameter of the zone of inhibition. Isolates that showed a resistant or intermediate phenotype were classified as resistant strains. *E. coli* ATCC 25,922 was used as the control strain.

### 4.4. Biofilm Collection, DNA Extraction, and Resistome Analysis

Five rocks per river site were collected and identified. After sterilisation, the rocks were placed in a net and deposited in the river at a location with little current. After 21 days of immersion, to allow for natural biofilm formation with the microbial communities of the river, the rocks were collected and carefully scraped with a sterile toothbrush, which was regularly rinsed with a small volume of sterile water [84,85]. The solution obtained was then centrifuged at 8000 × g for 10 min to remove excess water, and the resulting pellet was stored at –80 °C until analysis.

Water samples were collected at the same time as rocks to ensure monitoring of *E. coli* contamination rates at the sites.

Bacterial DNA from biofilms was extracted with a NucleoSpin Soil kit (Macherey–Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's protocol. DNA concentrations were measured with the Qubit instrument (Thermo Fisher Scientific, Waltham, MA USA). Some DNA from each sample was sent to the Biomics Platform C2RT (Institut Pasteur, Paris, France) for microbial community analysis and the remaining DNA was sent to the University of Limoges for the analysis of biofilm resistomes. The method used for resistome analysis was 96.96 BioMark® Dynamic Array for Real-Time PCR (Fluidigm Corporation, San Francisco, CA, USA). Threshold cycle (Ct) values were extracted with BioMark Real-Time PCR analysis software v1.2.0. Normalised gene abundance was calculated from 16S rRNA gene abundance according to the following formula: $2^{\{-[Ct(RG) - Ct(16S rRNA)]\}}$. The analytical procedure is described in more detail by Buelow [93,94].

### 4.5. Molecular Identification of Bacteria by 16S rRNA Gene Sequencing and Metagenomic Analysis

Amplification of the V3–V4 region of the 16S rRNA gene and MiSeq sequencing was performed by the Biomics platform of the Institut Pasteur Paris, France.

The V3–V4 variable region of the ribosomal DNA coding gene was amplified with the following prokaryote-specific universal primers: 341F (CCTACGGGNGGCWGCAG) and 785R (GACTACHVGGGTATCTAATCC) [95]. After Illumina sequencing, the raw data were cleaned with the “dada2” library [96] for use in R software v4.3.0 [97], which allows for filtering, merging, clustering, chimera removal, and taxonomy assignment (on the SILVA database [98]) of the amplicon sequence variants found. Data were analysed with “ggplot2” [99] and “phyloseq” [100] in order to generate diagrams according to the desired conditions. Statistical analyses were performed with the “vegan” library [101].

## 5. Conclusions

In Guadeloupe, rivers not affected by WWTP discharges have relatively low levels of ARB. Nevertheless, the frequent presence of *E. coli* in recreational freshwater, sometimes at concentrations that exceed regulatory standards, raises concern about the health of bathers. The analysis of bacterial communities at various sites showed similar compositions but dif-
ferent abundances, reflecting site-specific environmental conditions. The analysis of biofilm resistomes revealed traces of MGEs and ARGs, confirming limited antibiotic resistance in environments that do not receive human discharge, although clinical ARGs were identified. Better management of hot spots, such as those that receive effluents from WWTPs, tourist areas, and hospitals, should prevent the contamination of recreational areas by faecal bacteria and, consequently, help avoid the risk of dissemination of bacterial resistance.

**Author Contributions:** D.B.M. prepared the samples for the metagenomic studies and resistome analyses, generated some graphs, analysed all the data, and wrote the manuscript. E.C. conducted the collection campaigns and laboratory analyses of bacterial cultures, including antibiotic sensitivity analysis. C.D. supervised the characterisation of the resistome with the Fluidigm® PCR method and contributed to the development of the biofilm sampling protocol. I.Q. performed the bioinformatics analyses of the biofilm microbiome and generated the associated graphs. S.B. designed this study and revised the manuscript. S.G.-R. coordinated this study and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a FEDER grant (2018-FED-1084) financed by the European Union and the Guadeloupe Region (Programme Opérationnel FEDER-Guadeloupe-Conseil Régional 2014–2020). Biomics Platform, C2RT, Pasteur Institut, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA. D.B.M. was funded by a doctoral grant from the Agence Nationale de la Recherche (ANR-20-AMRB-0001-01). The funders had no role in the design of this study; in the collection, analysis or interpretation of the data; in writing the manuscript; or in the decision to publish the results.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** We thank Margaux Gashet for generating raw resistome data and for the graph of the associated relative abundance, Jean-Baptiste Courdeyre for his contribution to the sampling campaigns, Antoine Talarmin for manuscript revision, and Georges Michel Haustant and Laure Lemée (both from Biomics Platform, C2RT, Pasteur Institut, Paris, France) for technical support.

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

## References

1. Van Boeckel, T.P.; Brower, C.; Gilbert, M.; Grenfell, B.T.; Levin, S.A.; Robinson, T.P.; Teillant, A.; Laxminarayan, R. Global Trends in Antimicrobial Use in Food Animals. *Proc. Natl. Acad. Sci. USA* **2015**, *112*, 5649–5654. [CrossRef] [PubMed]

2. Paterson, D.L.; Bonomo, R.A. Extended-Spectrum Beta-Lactamases: A Clinical Update. *Clin. Microbiol. Rev.* **2005**, *18*, 657–686. [CrossRef] [PubMed]

3. Pal, C.; Asiani, K.; Arya, S.; Rensing, C.; Stekel, D.J.; Larsson, D.G.J.; Hobman, J.L. Chapter Seven—Metal Resistance and Its Association with Antibiotic Resistance. In *Advances in Microbial Physiology*; Poole, R.K., Ed.; Microbiology of Metal Ions; Academic Press: Cambridge, MA, USA, 2017; Volume 70, pp. 261–313.

4. Zheng, W.; Huyan, J.; Tian, Z.; Zhang, Y.; Wen, X. Clinical Class 1 Integron-Integrase Gene—A Promising Indicator to Monitor the Abundance and Elimination of Antibiotic Resistance Genes in an Urban Wastewater Treatment Plant. *Environ. Int.* **2020**, *135*, 105372. [CrossRef]

5. Gootz, T.D. The Global Problem of Antibiotic Resistance. *Crit. Rev. Immunol.* **2010**, *30*, 79–93. [CrossRef]

6. Prestinaci, F.; Pezzotti, P.; Pantosti, A. Antimicrobial Resistance: A Global Multifaceted Phenomenon. *Pathog. Glob. Health* **2015**, *109*, 309–318. [CrossRef] [PubMed]

7. Walsh, T.R.; Gales, A.C.; Laxminarayan, R.; Dodd, P.C. Antimicrobial Resistance: Addressing a Global Threat to Humanity. *PLOS Med.* **2023**, *20*, e1004264. [CrossRef] [PubMed]

8. Dereeper, A.; Gruel, G.; Pot, M.; Couvin, D.; Barbier, E.; Bastian, S.; Bambou, J.-C.; Gelu-Simeon, M.; Ferdinand, S.; Guyomard-Rabenirina, S.; et al. Limited Transmission of Klebsiella Pneumoniae among Humans, Animals, and the Environment in a Caribbean Island, Guadeloupe (French West Indies). *Microbiol. Spectr.* **2022**, *10*, e0124222. [CrossRef]

9. Bastian, S.; Nordmann, P.; Creton, E.; Malpote, E.; Thiery, G.; Martino, F.; Breurec, S.; Dortet, L. First Case of NDM-1 Producing Klebsiella Pneumoniae in Caribbean Islands. *Int. J. Infect. Dis. IJID Off. Publ. Int. Soc. Infect. Dis.* **2015**, *34*, 53–54. [CrossRef]

10. Breurec, S.; Bastian, S.; Cuzon, G.; Bernabeu, S.; Foucan, T.; Galanth, S.; Naas, T.; Dortet, L. Emergence of OXA-48-Producing Escherichia Coli in the Caribbean Islands. *J. Glob. Antimicrob. Resist.* **2015**, *3*, 217–218. [CrossRef]
11. Le Terrier, C.; Vinetti, M.; Bonjean, P.; Richard, R.; Jarrige, B.; Pons, B.; Madeux, B.; Piednoir, P.; Ardisson, F.; Elie, E.; et al. Impact of a Restrictive Antibiotic Policy on the Acquisition of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in an Endemic Region: A before-and-after, Propensity-Matched Cohort Study in a Caribbean Intensive Care Unit. *Crit. Care* **2021**, *25*, 261. [CrossRef]

12. Pot, M.; Reynaud, Y.; Couvin, D.; Dereeper, A.; Ferdinand, S.; Bastian, S.; Foucan, T.; Pommier, J.-D.; Valette, M.; Talarmin, A.; et al. Emergence of a Novel Lineage and Wide Spread of a blaCTX-M-15/IncHI2/ST1 Plasmid among Nosocomial Enterobacter in Guadeloupe. *Antibiotics* **2022**, *11*, 1443. [CrossRef]

13. Gruel, G.; Couvin, D.; Guyomard-Rabenirina, S.; Arlet, G.; Bambou, J.-C.; Pot, M.; Roy, X.; Talarmin, A.; Tressieres, B.; Ferdinand, S.; et al. High Prevalence of Bla CTXM-1/IncI1-Iγ/ST3 Plasmids in Extended-Spectrum β-Lactamase-Producing Escherichia Coli Isolates Collected From Domestic Animals in Guadeloupe (French West Indies). *Front. Microbiol.* **2022**, *13*, 882422. [CrossRef] [PubMed]

14. Sadikalay, S.; Reynaud, Y.; Guyomard-Rabenirina, S.; Falord, M.; Ducat, C.; Fabre, L.; Le Hello, S.; Talarmin, A.; Ferdinand, S. High Genetic Diversity of Extended-Spectrum β-Lactamases Producing *Escherichia coli* in Feces of Horses. *Vet. Microbiol.* **2018**, *219*, 117-122. [CrossRef] [PubMed]

15. Gruel, G.; Sellin, A.; Riveiro, H.; Pot, M.; Breurec, S.; Guyomard-Rabenirina, S.; Talarmin, A.; Ferdinand, S. Antimicrobial Use and Resistance in *Escherichia Coli* from Healthy Food-Producing Animals in Guadeloupe. *BMC Vet. Res.* **2021**, *17*, 116. [CrossRef] [PubMed]

16. Guyomard-Rabenirina, S.; Reynaud, Y.; Pot, M.; Albina, E.; Couvin, D.; Ducat, C.; Gruel, G.; Ferdinand, S.; Legreneur, P.; Le Hello, S.; et al. Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single blaCTX-M-1/IncI1/ST3 Plasmid Among Humans and Wild Animals. *Front. Microbiol.* **2020**, *11*, 1524. [CrossRef] [PubMed]

17. Pot, M.; Reynaud, Y.; Couvin, D.; Ducat, C.; Ferdinand, S.; Gravey, F.; Gruel, G.; Guérin, F.; Malpote, E.; Breurec, S.; et al. Wide Distribution and Specific Resistance Pattern to Third-Generation Cephalosporins of Enterobacter Cloacae Complex Members in Humans and in the Environment in Guadeloupe (French West Indies). *Front. Microbiol.* **2021**, *12*, 628058. [CrossRef] [PubMed]

18. Pot, M.; Guyomard-Rabenirina, S.; Couvin, D.; Ducat, C.; Enouf, V.; Ferdinand, S.; Gruel, G.; Malpote, E.; Talarmin, A.; Breurec, S.; et al. Dissemination of Extended-Spectrum-β-Lactamase-Producing Enterobacter Cloacae Complex from a Hospital to the Nearby Environment in Guadeloupe (French West Indies): ST114 Lineage Coding for a Successful IncHI2/ST1 Plasmid. *Antimicrob. Agents Chemother.* **2021**, *65*, e02146-20. [CrossRef]

19. Guyomard-Rabenirina, S.; Dartron, C.; Falord, M.; Sadikalay, S.; Ducat, C.; Richard, V.; Breurec, S.; Gros, O.; Talarmin, A. Resistance to Antimicrobial Drugs in Different Surface Waters and Wastewaters of Guadeloupe. *PLoS ONE* **2017**, *12*, e0173155. [CrossRef]

20. Rapport d'Activité 2019 de La Guadeloupe. 2020. Available online: https://www.iedom.fr/IMG/Pdf/Ra2019_guadeloupe_1_.pdf (accessed on 24 July 2023).

21. Ministère Chargé de la Santé Normes de Qualité Des Eaux et Classement Des Zones de Baignade. Available online: https://baignades.sante.gouv.fr/baignades/editorial/fr/controle/exemple.html (accessed on 28 December 2023).

22. Qualité Des Eaux de Baignade: Les Cartes 2020 de La Guadeloupe et Des Iles Du Nord—Qualité Des Eaux de Baignade En Guadeloupe—Sécurité Sanitaire—Protection Du Consommateur et Sécurité Alimentaire—Actions de l'État—Les Services de l'État En Guadeloupe. Available online: https://www.guadeloupe.gouv.fr/Actions-de-l-Etat/Protection-du-consommateur-et-securite-alimentaire/Securite-sanitaire/Qualite-des-eaux-de-baignade-en-Guadeloupe/Qualite-des-eaux-de-baignade-les-cartes-2020-de-la-Guadeloupe-et-des-Iles-du-Nord (accessed on 24 March 2023).

23. Servais, P.; Passerat, J. Antimicrobial Resistance of Fecal Bacteria in Waters of the Seine River Watershed (France). *Sci. Total Environ.* **2009**, *408*, 365-372. [CrossRef]

24. Watkinson, A.J.; Micalizzi, G.R.; Bates, J.R.; Costanzo, S.D. Novel Method for Rapid Assessment of Antibiotic Resistance in *Escherichia Coli* Isolates from Environmental Waters by Use of a Modified Chromogenic Agar. *Appl. Environ. Microbiol.* **2007**, *73*, 2224-2229. [CrossRef]

25. Hamelin, K.; Bruant, G.; El-Shaarawi, A.; Hill, S.; Edge, T.A.; Fairbrother, J.; Harel, J.; Maynard, C.; Masson, L.; Brousseau, R. Occurrence of Virulence and Antimicrobial Resistance Genes in *Escherichia coli* Isolates from Different Aquatic Ecosystems within the St. Clair River and Detroit River Areas. *Appl. Environ. Microbiol.* **2007**, *73*, 477-484. [CrossRef]

26. Liguori, K.; Keenum, I.; Davis, B.C.; Calarco, J.; Milligan, E.; Harwood, V.J.; Pruden, A. Antimicrobial Resistance Monitoring of Water Environments: A Framework for Standardized Methods and Quality Control. *Environ. Sci. Technol.* **2022**, *56*, 9149-9160. [CrossRef]

27. Proia, L.; von Schiller, D.; Sánchez-Melsió, A.; Sabater, S.; Borrego, C.M.; Rodríguez-Mozaz, S.; Balcázar, J.L. Occurrence and Persistence of Antibiotic Resistance Genes in River Biofilms after Wastewater Inputs in Small Rivers. *Environ. Pollut.* **2016**, *210*, 121-128. [CrossRef] [PubMed]

28. Donlan, R.M.; Costerton, J.W. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. *Clin. Microbiol. Rev.* **2002**, *15*, 167-193. [CrossRef] [PubMed]

29. Engemann, C.A.; Keen, P.L.; Knapp, C.W.; Hall, K.J.; Graham, D.W. Fate of Tetracycline Resistance Genes in Aquatic Systems: Migration from the Water Column to Peripheral Biofilms. *Environ. Sci. Technol.* **2008**, *42*, 5131-5136. [CrossRef]

30. Marti, E.; Jofre, J.; Balcazar, J.L. Prevalence of Antibiotic Resistance Genes and Bacterial Community Composition in a River Influenced by a Wastewater Treatment Plant. *PLoS ONE* **2013**, *8*, e78906. [CrossRef] [PubMed]
31. Pruden, A.; Pei, R.; Storteboom, H.; Carlson, K.H. Antibiotic Resistance Genes as Emerging Contaminants: Studies in Northern Colorado. *Environ. Sci. Technol.* **2006**, *40*, 7445–7450. [CrossRef] [PubMed]

32. Rodriguez-Mozaz, S.; Chamorro, S.; Marti, E.; Huerta, B.; Gros, M.; Sánchez-Melsió, A.; Borrego, C.M.; Barceló, D.; Balcázar, J.L. Occurrence of Antibiotics and Antibiotic Resistance Genes in Hospital and Urban Wastewaters and Their Impact on the Receiving River. *Water Res.* **2015**, *69*, 234–242. [CrossRef]

33. Anjum, M.F.; Schmitt, H.; Börjesson, S.; Berendonk, T.U.; Donner, E.; Stehling, E.G.; Boerlin, P.; Topp, E.; Jardine, C.; Li, X.; et al. The Potential of Using *E. coli* as an Indicator for the Surveillance of Antimicrobial Resistance (AMR) in the Environment. *Curr. Opin. Microbiol.* **2021**, *64*, 152–158. [CrossRef]

34. WHO. Integrated Global Surveillance on ESBL-Producing *E. coli* Using a "One Health" Approach: Implementation and Opportunities. Available online: https://www.who.int/publications-detail-redirect/9789240021402 (accessed on 16 September 2023).

35. Tornevi, A.; Bergstedt, O.; Forsberg, B. Precipitation Effects on Microbial Pollution in a River: Lag Structures and Seasonal Effect Modification. *PLoS ONE* **2014**, *9*, e98546. [CrossRef]

36. Baudart, J.; Paniel, N. Sources et Devenir Des Micro-Organismes Pathogènes Dans Les Environnements Aquatiques. *Rev. Francoph. Lab.* **2014**, *2014*, 29-39. [CrossRef] [PubMed]

37. Ebmeyer, S.; Kristiansson, E.; Larsson, D.G.J. A Framework for Identifying the Recent Origins of Mobile Antibiotic Resistance Genes. *Commun. Biol.* **2021**, *4*, 8. [CrossRef] [PubMed]

38. Yashiro, E.; McManus, P.S. Effect of Streptomycin Treatment on Bacterial Community Structure in the Apple Phyllosphere. *PLoS ONE* **2012**, *7*, e37131. [CrossRef] [PubMed]

39. Blaak, H.; van Hoek, A.H.A.M.; Hamidjaja, R.A.; van der Plaats, R.Q.J.; Kerkhof-de Heer, L.; de Roda Husman, A.M.; Schets, F.M. Distribution, Numbers, and Diversity of ESBL-Producing *E. coli* in the Poultry Farm Environment. *PLoS ONE* **2015**, *10*, e0135402. [CrossRef]

40. Daly, S.M.; Sturge, C.R.; Felder-Scott, C.F.; Geller, B.L.; Greenberg, D.E. MCR-1 Inhibition with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Sensitivity to Polymyxin in *Escherichia coli*. *mBio* **2017**, *8*, e01315-17. [CrossRef] [PubMed]

41. Sun, J.; Xu, Y.; Gao, R.; Lin, J.; Wei, W.; Srinivas, S.; Li, D.; Yang, R.-S.; Li, X.-P.; Liao, X.-P.; et al. Deciphering MCR-2 Colistin Resistance. *mBio* **2017**, *8*, e00625-17. [CrossRef]

42. Deng, Y.; Bao, X.; Ji, L.; Chen, L.; Liu, J.; Miao, J.; Chen, D.; Bian, H.; Li, Y.; Yu, G. Resistance Integrons: Class 1, 2 and 3 Integrons. *Ann. Clin. Microbiol. Antimicrob.* **2015**, *14*, 45. [CrossRef]

43. Uyaguari-Díaz, M.I.; Croxen, M.A.; Luo, Z.; Cronin, K.I.; Chan, M.; Baticados, W.N.; Nesbitt, M.J.; Li, S.; Miller, K.M.; Dooley, D.; et al. Human Activity Determines the Presence of Integron-Associated and Antibiotic Resistance Genes in Southwestern British Columbia. *Front. Microbiol.* **2018**, *9*, 852. [CrossRef]

44. Bourdonnais, E.; Colcanap, D.; Le Bris, C.; Brauge, T.; Midelet, G. Occurrence of Indicator Genes of Antimicrobial Resistance Contamination in the English Channel and North Sea Sectors and Interactions with Environmental Variables. *Front. Microbiol.* **2022**, *13*, 883081. [CrossRef]

45. Baker-Austin, C.; Wright, M.S.; Stepanauskas, R.; McArthur, J.V. Co-Selection of Antibiotic and Metal Resistance. *Trends Microbiol.* **2006**, *14*, 176-182. [CrossRef]

46. Larsson, D.G.J.; Flach, C.-F. Antibiotic Resistance in the Environment. *Nat. Rev. Microbiol.* **2022**, *20*, 257-269. [CrossRef] [PubMed]

47. Seiler, C.; Berendonk, T. Heavy Metal Driven Co-Selection of Antibiotic Resistance in Soil and Water Bodies Impacted by Agriculture and Aquaculture. *Front. Microbiol.* **2012**, *3*, 399. [CrossRef] [PubMed]

48. Siedlecka, A.; Wolf-Baca, M.J.; Piekarska, K. Antibiotic and Disinfectant Resistance in Tap Water Strains—Insight into the Resistance of Environmental Bacteria. *Pol. J. Microbiol.* **2021**, *70*, 57–67. [CrossRef] [PubMed]

49. Zhu, S.; Yang, B.; Jia, Y.; Yu, F.; Wang, Z.; Liu, Y. Comprehensive Analysis of Disinfectants on the Horizontal Transfer of Antibiotic Resistance Genes. *J. Hazard. Mater.* **2023**, *453*, 131428. [CrossRef] [PubMed]

50. Barberán, A.; Casamayor, E. Global Phylogenetic Community Structure and β-Diversity Patterns in Surface Bacterioplankton Metacommunities. *Aquat. Microb. Ecol.* **2010**, *59*, 1–10. [CrossRef]

51. Bartrons, M.; Catalan, J.; Casamayor, E.O. High Bacterial Diversity in Epilithic Biofilms of Oligotrophic Mountain Lakes. *Microb. Ecol.* **2012**, *64*, 860–869. [CrossRef] [PubMed]

52. Battin, T.J.; Besemer, K.; Bengtsson, M.M.; Romani, A.M.; Packmann, A.I. The Ecology and Biogeochemistry of Stream Biofilms. *Nat. Rev. Microbiol.* **2016**, *14*, 251–263. [CrossRef]

53. Newton A Guide to the Natural History of Freshwater Lake Bacteria. Available online: https://journals.asm.org/doi/epdf/10.128/mmbr.00028-10?src=getftr (accessed on 24 July 2023).

54. Pineda-Mora, D.; Juárez-López, A.L.; Toribio-Jiménez, J.; Leal-Ascencio, M.T.; Ruvalcaba-Ledezma, J.C.; Castelán-Sánchez, H.G.; Aguirre-Noyola, J.L.; Arp, P.A. Diversity and Functions of Epilithic Riverine Biofilms. *Water. Air. Soil Pollut.* **2020**, *231*, 391. [CrossRef]

55. Wang, L.; Zhang, J.; Li, H.; Yang, H.; Peng, C.; Peng, Z.; Lu, L. Shift in the Microbial Community Composition of Surface Water and Sediment along an Urban River. *Sci. Total Environ.* **2018**, *627*, 600–612. [CrossRef]

56. Araya, R.; Tani, K.; Takagi, T.; Yamaguchi, N.; Nasu, M. Bacterial Activity and Community Composition in Stream Water and Biofilm from an Urban River Determined by Fluorescent in Situ Hybridization and DGGE Analysis. *FEMS Microbiol. Ecol.* **2003**, *43*, 111–119. [CrossRef]
57. Betiku, O.C.; Sarjeant, K.C.; Ngatia, L.W.; Aghimien, M.O.; Odewumi, C.O.; Latinwo, L.M. Evaluation of Microbial Diversity of Three Recreational Water Bodies Using 16S rRNA Metagenomic Approach. *Sci. Total Environ.* **2021**, *771*, 144773. [CrossRef] [PubMed]

58. Burkert, U.; Warnecke, F.; Babenzien, D.; Zwirnmann, E.; Pernthaler, J. Members of a Readily Enriched Beta-Proteobacterial Clade Are Common in Surface Waters of a Humic Lake. *Appl. Environ. Microbiol.* **2003**, *69*, 6550–6559. [CrossRef] [PubMed]

59. Jin, D.; Kong, X.; Cui, B.; Jin, S.; Xie, Y.; Wang, X.; Deng, Y. Bacterial Communities and Potential Waterborne Pathogens within the Typical Urban Surface Waters. *Sci. Rep.* **2018**, *8*, 13368. [CrossRef] [PubMed]

60. Vega, L.; Jaimes, J.; Morales, D.; Martínez, D.; Cruz-Saavedra, L.; Muñoz, M.; Ramírez, J.D. Microbial Communities' Characterization in Urban Recreational Surface Waters Using Next Generation Sequencing. *Microb. Ecol.* **2021**, *81*, 847–863. [CrossRef]

61. Zhang, J.; Yang, Y.; Zhao, L.; Li, Y.; Xie, S.; Liu, Y. Distribution of Sediment Bacterial and Archaeal Communities in Plateau Freshwater Lakes. *Appl. Microbiol. Biotechnol.* **2015**, *99*, 3291–3302. [CrossRef]

62. Andrzejak, T.; Raje, H.; LaFleur, G.; Willis, J.; Boopathy, R. Water Quality and Antibiotic Resistance in the Recreational Waters. *Bioresour. Technol.* **2023**, *370*, 128546. [CrossRef] [PubMed]

63. Ragon, M.; Fontaine, M.C.; Moreira, D.; López-García, P. Different Biogeographic Patterns of Prokaryotes and Microbial Eukaryotes in Epilithic Biofilms. *Mol. Ecol.* **2012**, *21*, 3852–3868. [CrossRef] [PubMed]

64. Antunes, J.T.; Sousa, A.G.G.; Azevedo, J.; Rego, A.; Leão, P.N.; Vasconcelos, V. Distinct Temporal Succession of Bacterial Communities in Early Marine Biofilms in a Portuguese Atlantic Port. *Front. Microbiol.* **2020**, *11*, 1938. [CrossRef]

65. Fierer, N.; Bradford, M.A.; Jackson, R.B. Toward an Ecological Classification of Soil Bacteria. *Ecology* **2007**, *88*, 1354–1364. [CrossRef]

66. Ponsatí, L.; Corcoll, N.; Petrović, M.; Picó, Y.; Ginebreda, A.; Tornés, E.; Guasch, H.; Barceló, D.; Sabater, S. Multiple-Stressor Effects on River Biofilms under Different Hydrological Conditions. *Freshw. Biol.* **2016**, *61*, 2102–2115. [CrossRef]

67. Zoppini, A.; Amalfitano, S.; Fazi, S.; Puddu, A. Dynamics of a Benthic Microbial Community in a Riverine Environment Subject to Hydrological Fluctuations (Mulargia River, Italy). In *Global Change and River Ecosystems—Implications for Structure, Function and Ecosystem Services*; Stevenson, R.J., Sabater, S., Eds.; Developments in Hydrobiology 215; Springer: Dordrecht, The Netherlands, 2010; pp. 37–51, ISBN 978-94-007-0608-8.

68. Bauer, M.; Kube, M.; Teeling, H.; Richter, M.; Lombardot, T.; Allers, E.; Würdemann, C.A.; Quast, C.; Kuhl, H.; Knaust, F.; et al. Whole Genome Analysis of the Marine Bacteroidetes' Gramella Forsetii' Reveals Adaptations to Degradation of Polymeric Organic Matter. *Environ. Microbiol.* **2006**, *8*, 2201–2213. [CrossRef]

69. Wang, J.; Jenkins, C.; Webb, R.I.; Fuerst, J.A. Isolation of Gemmata-Like and Isosphaera-Like Planctomycete Bacteria from Soil and Freshwater. *Appl. Environ. Microbiol.* **2002**, *68*, 417–422. [CrossRef] [PubMed]

70. Paerl, H.W.; Hall, N.S.; Calandrino, E.S. Controlling Harmful Cyanobacterial Blooms in a World Experiencing Anthropogenic and Climatic-Induced Change. *Sci. Total Environ.* **2011**, *409*, 1739–1745. [CrossRef]

71. Garrido-Oter, R.; Nakano, R.T.; Dombrowski, N.; Ma, K.-W.; AgBiome Team; McHardy, A.C.; Schulze-Lefert, P. Modular Traits of the Rhizobiales Root Microbiota and Their Evolutionary Relationship with Symbiotic Rhizobia. *Cell Host Microbe* **2018**, *24*, 155–167.e5. [CrossRef]

72. Lepleux, C.; Turpault, M.P.; Oger, P.; Frey-Klett, P.; Uroz, S. Correlation of the Abundance of Betaproteobacteria on Mineral Surfaces with Mineral Weathering in Forest Soils. *Appl. Environ. Microbiol.* **2012**, *78*, 7114–7119. [CrossRef]

73. Kondrotaite, Z.; Valk, L.C.; Petriglieri, F.; Singleton, C.; Nierychlo, M.; Dueholm, M.K.D.; Nielsen, P.H. Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems. *Front. Microbiol.* **2022**, *13*, 917553. [CrossRef] [PubMed]

74. McIlroy, S.J.; Nielsen, P.H. The Family Saprospiraceae. In *The Prokaryotes: Other Major Lineages of Bacteria and The Archaea*; Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson, F., Eds.; Springer: Berlin/Heidelberg, Germany, 2014; pp. 863–889, ISBN 978-3-642-38954-2.

75. Marín, A.; Feijoo, P.; de Llanos, R.; Carbonetto, B.; González-Torres, P.; Tena-Medialdea, J.; García-March, J.R.; Gámez-Pérez, J.; Cabedo, L. Microbiological Characterization of the Biofilms Colonizing Bioplastics in Natural Marine Conditions: A Comparison between PHBV and PLA. *Microorganisms* **2023**, *11*, 1461. [CrossRef] [PubMed]

76. Lyautey, E.; Jackson, C.R.; Cayrou, J.; Rols, J.-L.; Garabétian, F. Bacterial Community Succession in Natural River Biofilm Assemblages. *Microb. Ecol.* **2005**, *50*, 589–601. [CrossRef]

77. Chen, H.; Zhang, S.; Lv, X.; Guo, S.; Ma, Y.; Han, B.; Hu, X. Interactions between Suspended Sediments and Submerged Macrophytes-Epiphytic Biofilms under Water Flow in Shallow Lakes. *Water Res.* **2022**, *222*, 118911. [CrossRef]

78. Ge, Z.; Ma, Z.; Hong, W.; Liu, K.; Yan, S.; Song, W.; Zhang, J. Temporal Variations in Reactive Oxygen Species in Biofilms of Submerged Macrophytes: The Key Role of Microbial Metabolism Mediated by Oxygen Fluctuations. *J. Hazard. Mater.* **2024**, *461*, 132542. [CrossRef]

79. Kowalczyk, A.; Price, O.R.; van der Gast, C.J.; Finnegan, C.J.; van Egmond, R.A.; Schäfer, H.; Bending, G.D. Spatial and Temporal Variability in the Potential of River Water Biofilms to Degrade P-Nitrophenol. *Chemosphere* **2016**, *164*, 355–362. [CrossRef]

80. Kelly, J.J.; Minalt, N.; Culotti, A.; Pryor, M.; Packman, A. Temporal Variations in the Abundance and Composition of Biofilm Communities Colonizing Drinking Water Distribution Pipes. *PLoS ONE* **2014**, *9*, e98542. [CrossRef]

81. Zancarini, A.; Echenique-Subiabre, I.; Debroas, D.; Taïb, N.; Quiblier, C.; Humbert, J.-F. Deciphering Biodiversity and Interactions between Bacteria and Microeukaryotes within Epilithic Biofilms from the Loue River, France. *Sci. Rep.* **2017**, *7*, 4344. [CrossRef]
82. Kaevska, M.; Videnska, P.; Sedlar, K.; Slana, I. Seasonal Changes in Microbial Community Composition in River Water Studied Using 454-Pyrosequencing. Springer Plus **2016**, *5*, 409. [CrossRef]

83. Wang, J.; Fan, H.; He, X.; Zhang, F.; Xiao, J.; Yan, Z.; Feng, J.; Li, R. Response of Bacterial Communities to Variation in Water Quality and Physicochemical Conditions in a River-Reservoir System. Glob. Ecol. Conserv. **2021**, *27*, e01541. [CrossRef]

84. Matviichuk, O.; Mondamert, L.; Geffroy, C.; Gaschet, M.; Dagot, C.; Labanowski, J. River Biofilms Microbiome and Resistome Responses to Wastewater Treatment Plant Effluents Containing Antibiotics. Front. Microbiol. **2022**, *13*, 795206. [CrossRef] [PubMed]

85. Aubertheau, E.; Stalder, T.; Mondamert, L.; Ploy, M.-C.; Dagot, C.; Labanowski, J. Impact of Wastewater Treatment Plant Discharge on the Contamination of River Biofilms by Pharmaceuticals and Antibiotic Resistance. Sci. Total Environ. **2017**, *579*, 1387–1398. [CrossRef] [PubMed]

86. Lin, Q.; Sekar, R.; Marrs, R.; Zhang, Y. Effect of River Ecological Restoration on Biofilm Microbial Community Composition. Water **2019**, *11*, 1244. [CrossRef]

87. Harnisz, M.; Kiedrzyńska, E.; Kiedrzyński, M.; Korzeniewska, E.; Czatzkowska, M.; Koniuszewska, I.; Jóźwik, A.; Szklarek, S.; Niestępski, S.; Zalewski, M. The Impact of WWTP Size and Sampling Season on the Prevalence of Antibiotic Resistance Genes in Wastewater and the River System. Sci. Total Environ. **2020**, *741*, 140466. [CrossRef]

88. ISO 9308-3:1998(Fr); Qualité de l'eau—Recherche et Dénombrement Des *Escherichia coli* et Des Bactéries Coliformes—Partie 3: Méthode Miniaturisée (Nombre Le plus Probable) Pour La Recherche et Le Dénombrement Des *E. coli* Dans Les Eaux de Surface et Résiduaires. Plateforme de consultation en ligne (OBP). Available online: https://www.iso.org/obp/ui/fr/#iso:std:iso:9308:-3:ed-1:v1:fr (accessed on 12 August 2023).

89. European Parliament, Council of the European Union. Directive 2006/7/EC of the European Parliament and of the Council of 15 February 2006 Concerning the Management of Bathing Water Quality and Repealing Directive 76/160/EEC. Off. J. Eur. Union **2006**, *64*, 37–51.

90. Chapitre II: Piscines et Baignades. (Articles L1332-1 à L1332-9)—Légifrance. Available online: https://www.legifrance.gouv.fr/codes/id/LEGISCTA000006171063 (accessed on 12 September 2023).

91. Section 2: Règles Sanitaires Applicables Aux Eaux de Baignade (Articles D1332-14 à D1332-38-1)—Légifrance. Available online: https://www.legifrance.gouv.fr/codes/id/LEGISCTA000019506596 (accessed on 12 September 2023).

92. La Société Française de Microbiologie. CASFM/EUCAST AVRIL 2021 V1.0. 2021. Available online: https://www.sfm-microbiologie.org/2021/04/23/casfm-avril-2021-v1-0/ (accessed on 12 September 2023).

93. Buelow, E.; Bayjanov, J.R.; Majoor, E.; Willems, R.J.; Bonten, M.J.; Schmitt, H.; van Schaik, W. Limited Influence of Hospital Wastewater on the Microbiome and Resistome of Wastewater in a Community Sewerage System. FEMS Microbiol. Ecol. **2018**, *94*, fiy087. [CrossRef]

94. Buelow, E.; Rico, A.; Gaschet, M.; Lourenço, J.; Kennedy, S.P.; Wiest, L.; Ploy, M.-C.; Dagot, C. Hospital Discharges in Urban Sanitation Systems: Long-Term Monitoring of Wastewater Resistome and Microbiota in Relationship to Their Eco-Exposome. Water Res. X **2020**, *7*, 100045. [CrossRef] [PubMed]

95. Beckers, B.; Op De Beeck, M.; Thijs, S.; Truyens, S.; Weyens, N.; Boerjan, W.; Vangronsveld, J. Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies. Front. Microbiol. **2016**, *7*, 650. [CrossRef]

96. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-Resolution Sample Inference from Illumina Amplicon Data. Nat. Methods **2016**, *13*, 581–583. [CrossRef] [PubMed]

97. Ihaka, R.; Gentleman, R. R: A Language for Data Analysis and Graphics. J. Comput. Graph. Stat. **1996**, *5*, 299–314. [CrossRef]

98. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glöckner, F.O. The SILVA Ribosomal RNA Gene Database Project: Improved Data Processing and Web-Based Tools. Nucleic Acids Res. **2013**, *41*, D590–D596. [CrossRef] [PubMed]

99. Wickham, H. Ggplot2: Use R! Springer International Publishing: Cham, Switzerland, 2016; ISBN 978-3-319-24275-0.

100. McMurdie, P.J.; Holmes, S. Phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. PLoS ONE **2013**, *8*, e61217. [CrossRef]

101. Oksanen, J.; Kindt, R.; Legendre, P.; O'Hara, B.; Stevens, M.H.H.; Oksanen, M.J.; Suggests, M. The Vegan Package. Community Ecol. Package **2007**, *10*, 719.

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