# Carbapenemase-producing Gram-negative bacteria in aquatic environments: a review

Zineb Cherak, Lotfi Loucif, Abdelhamid Moussi, Jean-Marc Rolain

► To cite this version:

Zineb Cherak, Lotfi Loucif, Abdelhamid Moussi, Jean-Marc Rolain. Carbapenemase-producing Gram-negative bacteria in aquatic environments: a review. Journal of Global Antimicrobial Resistance, 2021, 25, pp.287-309. 10.1016/j.jgar.2021.03.024. hal-03333771

HAL Id: hal-03333771

https://amu.hal.science/hal-03333771

Submitted on 10 Sep 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Carbapenemase-producing Gram-negative bacteria in aquatic environments: a review

<figure><img src="image_3.png" /></figure>

Zineb Cherakª, Lotfi Loucifᵇ,*, Abdelhamid Moussiª, Jean-Marc Rolainᶜ,ᵈ

## ARTICLE INFO

**Article history:**

Received 10 November 2020

Revised 4 March 2021

Accepted 20 March 2021

Available online 23 April 2021

Editor: Dr Jon Hobman

**Keywords:**

Carbapenemase

Gram-negative bacilli

Aquatic environment

Epidemiology

## ABSTRACT

Antibiotic resistance is one of the greatest public-health challenges worldwide, especially with regard to Gram-negative bacteria (GNB). Carbapenems are the β-lactam antibiotics of choice with the broadest spectrum of activity and, in many cases, are the last-resort treatment for several bacterial infections. Carbapenemase-encoding genes, mainly carried by mobile genetic elements, are the main mechanism of resistance against carbapenems in GNB. These enzymes exhibit a versatile hydrolytic capacity and confer resistance to most β-lactam antibiotics. After being considered a clinical issue, increasing attention is being given to the dissemination of such resistance mechanisms in the environment and especially through water. Aquatic environments are among the most significant microbial habitats on our planet, known as a favourable medium for antibiotic gene transfer, and they play a crucial role in the huge spread of drug resistance in the environment and the community. In this review, we present current knowledge regarding the spread of carbapenemase-producing isolates in different aquatic environments, which may help the implementation of control and prevention strategies against the spread of such dangerous resistant agents in the environment.

© 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

## 1. Introduction

Antibiotic resistance is one of the greatest public-health challenges worldwide, especially with regard to Gram-negative bacteria (GNB). In 2016, the World Health Organization (WHO) was called on by its member states to name a priority list of drug-resistant bacteria that require the development of new effective medicines. Interestingly, the critical priority level was reserved for carbapenem-resistant *Acinetobacter baumannii*, *Pseudomonas aeruginosa* and Enterobacteriaceae as well as third-generation cephalosporin-resistant Enterobacteriaceae [1]. Carbapenems are the β-lactam antibiotics with the broadest spectrum of activity. Imipenem, first marketed in 1985, was the first carbapenem available for the treatment of bacterial infections [2]. Thereafter, several carbapenems were developed in the subsequent two decades.

Nowadays, the most clinically used carbapenems are ertapenem, meropenem, doripenem and imipenem [3]. However, in less than a decade after their use, carbapenem-resistant GNB have emerged [4]. Owing to the fact that carbapenems are the antibiotics of choice and, in many cases, the last-resort treatment for several bacterial infections, the emergence and spread of carbapenem-resistant GNB are currently a major global public-health crisis [5]. Carbapenem resistance in GNB may be conferred by various mechanisms, including quantitative and/or qualitative changes in membrane permeability owing to chromosomal mutations in efflux pump-encoding genes or alterations in the expression and/or function of porins, the association of impermeability with extended-spectrum β-lactamases (ESBLs) or overexpression of AmpC β-lactamases or by the production of carbapenem-hydrolysing enzymes ('carbapenemases'), which represent the most worrying mechanism of carbapenem resistance. These enzymes exhibit a versatile hydrolytic capacity and confer resistance to most β-lactam antibiotics [2,3,6-8].

**Article history:**

Received 10 November 2020

Revised 4 March 2021

Accepted 20 March 2021

Available online 23 April 2021

Editor: Dr Jon Hobman

**Keywords:**

Carbapenemase

Gram-negative bacilli

Aquatic environment

Epidemiology

<footnote>ªLaboratoire de Génétique, Biotechnologie et Valorisation des Bio-ressources (GBVB), Faculté des Sciences Exactes et des Sciences de la Nature et de la Vie, Université Mohamed Khider, Biskra, Algeria</footnote>

<footnote>ᵇLaboratoire de Biotechnologie des Molécules Bioactives et de la Physiopathologie Cellulaire (LBMBPC), Département de Microbiologie et de Biochimie, Faculté des Sciences de la Nature et de la Vie, Université de Batna 2, Batna, Algeria</footnote>

<footnote>ᶜAix-Marseille Université, IRD, MEPHI, Faculté de Médecine et de Pharmacie, Marseille, France</footnote>

<footnote>ᵈIHU Méditerranée Infection, Marseille, France; and Assistance Publique des Hôpitaux de Marseille, Marseille, France</footnote>

<footnote>* Corresponding author. Tel.: +213 5 40 92 54 00. E-mail address: lotfiloucif@hotmail.fr (L. Loucif).</footnote>
Classically, antibiotic resistance has been known to be restricted to clinical settings [9]. However, several studies have demonstrated the dissemination of resistant organisms in the environment, particularly in water. Indeed, water is one of the most significant microbial habitats on our planet and it has been proven that antibiotic resistance genes are common in different water ecosystems, which may play a crucial role in the propagation of antibiotic resistance between the natural environment and humans and other animals [10-12].

In this review, we present current knowledge regarding the spread of carbapenemase-producing isolates in different aquatic environments, which may help the implementation of control and prevention strategies against the spread of such dangerous resistant agents in the environment.

For this purpose, we carried out a comprehensive literature search on PubMed and Google Scholar websites. We included papers published in English language up to April 2020 using the following search terms and/or phrases: 'Gram negative bacilli', 'Enterobacteriaceae', 'Pseudomonas aeruginosa', 'Acinetobacter baumannii', and 'carbapenemases', 'metallo-β-lactamases', 'KPC', 'GES', 'IMI', 'VIM', 'NDM', 'IMP', 'OXA-48', and 'water environments', 'aquatic environments', 'water', 'wastewater', 'sewage', 'hospital wastewater', 'hospital sewage', 'wastewater treatment plants', 'surface water', 'ground water'. Search terms were separated by the 'AND' Boolean operator.

## 2. Carbapenemases in Gram-negative bacteria

Carbapenemases constitute a large variety of enzymes that are categorised either functionally (Bush classification) or genetically (Ambler molecular classification) [5]. However, the most common classification is the molecular one based on the Ambler classification scheme [13] in which carbapenemases are assigned to three of the four Ambler classes (A, B and D). According to the functional classification, they fall under the functional groups 2df, 2f, 3a and 3b [14]. Despite the great number of carbapenemase enzymes identified in GNB, the five major and most prevalent carbapenemases are KPC (*Klebsiella pneumoniae* carbapenemase), NDM (New Delhi metallo-β-lactamase), IMP (imipenem-resistant *Pseudomonas*), VIM (Verona integron-encoded metallo-β-lactamase) and OXA-48 (oxacillinase) [15,16]. Carbapenemases are either chromosomally encoded, such as SME (*Serratia marcescens* enzyme) and NMC-A (non-metallo-carbapenemase-A), which are usually reported on the chromosome of some Enterobacterales species including *S. marcescens* and *Enterobacter cloacae*, or more frequently are encoded by mobile genetic elements including plasmids, integrons and transposons [5,17].

## 3. Epidemiology of carbapenemase-producing Gram-negative bacteria in aquatic environments

Carbapenemases of the three Ambler classes have been detected in aquatic environments whether using culture or culture-independent methods. The worldwide epidemiology of carbapenemase-producers isolated from various aquatic environments is summarised in Fig. 1 and presented below by Ambler class.

### 3.1. Class A carbapenemases

The first reported class A carbapenem-hydrolysing β-lactamase (SME) was from the UK in a *S. marcescens* isolate in 1990 [3]. Enzymes in this β-lactamase class are serine proteins (using a serine residue for their activity) and are inhibited by clavulanic acid, tazobactam and boronic acid compounds [6,18]. They have a broad

spectrum of activity including penicillins, cephalosporins, aztreonam and carbapenems [18]. The most prevalent and clinically significant enzyme among the class A carbapenemases is KPC, which is commonly identified in Enterobacterales species but also occasionally in *P. aeruginosa* and *A. baumannii* [13,19].

#### 3.1.1. Enterobacterales

Class A carbapenemase-producing Enterobacterales have been widely isolated from aquatic environments worldwide (Table 1; Fig. 2). In 2004, Henriques et al. characterised a new class A carbapenemase-encoding gene designated as SFC-1 (for *Serratia fonticola* carbapenemase) on the chromosome of a *S. fonticola* isolate obtained from untreated drinking water in Portugal [20]. Furthermore, IMI-2-producing *Enterobacter asburiae* isolates were recovered from rivers and lake water in the USA and France [21–23]. In addition, Piedra-Carrasco et al. have reported the isolation of IMI-2-producing *E. cloacae* from river water in Spain [24]. Another Ambler class A enzyme that is commonly reported among members of the Enterobacterales is KPC, whose variants are the most detected class A carbapenemases in Enterobacterales isolated from aquatic environments. KPC-producing *Enterobacter* spp., *Klebsiella* spp., *Citrobacter* spp., *Kluyvera* spp., *Escherichia coli*, *E. cloacae*, *Enterobacter* kobei, *E. asburiae*, *K. pneumoniae*, *Klebsiella oxytoca* and *Citrobacter freundii* complex have been isolated from river water, seawater, hospital sewage, wastewater treatment plants (WWTPs), drinking water and a hospital water dispenser [25–35]. KPC-2-producing *Citrobacter* spp., *Enterobacter* spp., *Klebsiella* spp., *Serratia* spp., *Raoultella* spp., *Kluyvera* spp., *Shigella* spp., *Escherichia* spp., *E. coli*, *K. pneumoniae*, *Klebsiella quasipneumoniae*, *K. oxytoca*, *E. cloacae* complex, *E. cloacae*, *E. kobei*, *E. asburiae*, *C. freundii*, *Citrobacter braakii*, *Citrobacter farmeri*, *Kluyvera georgiana*, *Kluyvera ascorbata*, *Kluyvera cryocrescens*, *Raoultella ornithinolytica*, *Raoultella planticola* and *Raoultella terrigena* have been isolated from rivers, hospital sewage, WWTPs, seawater and wells [24,36–57]. Another KPC variant, namely KPC-3, has been detected in *K. pneumoniae* isolated from wells and WWTPs in Italy [45,58], in *K. pneumoniae* cultivated from a Portuguese river [59], and in *E. coli* and *C. freundii* isolates obtained from WWTPs in the USA [57]. In addition, the new variant KPC-26 was first identified in *Klebsiella* spp. and *Enterobacter* spp. isolates obtained from seawater in Brazil [48]. Other Ambler class A enzymes that have been shown to possess carbapenemase activity are some GES variants (for Guiana extended spectrum). The GES-5 enzyme has been detected in *K. pneumoniae* isolated from stream water [60], in *R. ornithinolytica* and *Citrobacter* sp. recovered from river water [57,59], in *Enterobacter* spp. isolates obtained from seawater [25,48], in *Citrobacter* spp., *E. coli*, *K. pneumoniae*, *K. oxytoca* and *E. cloacae* obtained from hospital sewage [53,61,62], in *E. cloacae* complex, in *K. pneumoniae* and in *R. ornithinolytica* isolated from WWTPs [53,57,63]. GES-6-producing *Citrobacter* spp., *E. coli* and *K. quasipneumoniae* have been isolated from hospital sewage in Taiwan [53]. In addition, the GES-16 enzyme has been detected in *Enterobacter* spp. and *Klebsiella* spp. isolates recovered from seawater [25,48] and in *K. pneumoniae* obtained from river water [42]. GES-20-producing *K. oxytoca* and *E. kobei* were isolated from river water in the Philippines [64]. Finally, a GES-24-producing *Klebsiella variicola* was isolated from a WWTP in Japan [53].

#### 3.1.2. Other Gram-negative bacilli

Compared with Enterobacterales, few published reports have documented the isolation of class A carbapenemase-producing glucose-non-fermenting GNB from aqueous ecosystems. KPC-, KPC-2-, GES-5- and GES-16-producing *Acinetobacter* spp. and *Aeromonas* spp. isolates were recovered from WWTPs, hospital sewage, and river and seawater samples in the USA, Brazil and China [25,29,40,43,48,65]. In addition, the GES-31 carbapenemase was first described in an *Aeromonas punctata* isolate recovered from a
<table><caption>**Table 1**
Epidemiology of class A carbapenemase-producers detected in aquatic environments</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">GES-5</td><td rowspan="3">Brazil</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (1)<br/>Klebsiella oxytoca (1)</td><td>-</td><td>-</td><td>[62]</td></tr><tr><td rowspan="2">Seawater</td><td>Enterobacter (2)</td><td>-</td><td>-</td><td>[25]</td></tr><tr><td>Aeromonas (1), Acinetobacter (1)</td><td>-</td><td>-</td><td>[48]</td></tr><tr><td rowspan="3">Japan</td><td rowspan="3">WWTP</td><td>Raoultella ornithinolytica (1)</td><td>-</td><td>-</td><td>[53]</td></tr><tr><td>Klebsiella pneumoniae (1)</td><td>ST2791</td><td>-</td><td>-</td></tr><tr><td>Citrobacter sp. (1)</td><td>-</td><td>Inc3-16</td><td>[59]</td></tr><tr><td rowspan="2">Portugal</td><td>River</td><td>Klebsiella pneumoniae (4)</td><td>ST961</td><td>-</td><td>[60]</td></tr><tr><td>Stream water</td><td>Klebsiella pneumoniae (11)</td><td>ST11, ST15, ST19, ST16, ST844, ST2791, ST2785</td><td>-</td><td>[53]</td></tr><tr><td rowspan="3">Taiwan</td><td rowspan="3">Hospital sewage</td><td>Enterobacter cloacae (1)</td><td>ST928</td><td>-</td><td>-</td></tr><tr><td>Escherichia coli (6)</td><td>ST744, ST49</td><td>-</td><td>-</td></tr><tr><td>Citrobacter (1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="3">UK</td><td rowspan="3">Hospital sewage</td><td>Klebsiella oxytoca (6)</td><td>-</td><td>-</td><td>[61]</td></tr><tr><td>Enterobacter cloacae complex (5)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Klebsiella pneumoniae (1)</td><td>-</td><td>-</td><td>[63]</td></tr><tr><td rowspan="3">USA</td><td>River</td><td>Raoultella ornithinolytica (1)</td><td>-</td><td>-</td><td>[57]</td></tr><tr><td rowspan="2">WWTP</td><td>Enterobacter cloacae complex (1)</td><td>ST595</td><td>-</td><td>-</td></tr><tr><td>Escherichia coli (1)</td><td>ST540</td><td>-</td><td>[53]</td></tr><tr><td rowspan="2">Taiwan</td><td rowspan="2">Hospital sewage</td><td>Klebsiella quasipneumoniae (5)</td><td>ST1584, ST367</td><td>-</td><td>-</td></tr><tr><td>Citrobacter (1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="2">GES-7 (BIC-1)</td><td rowspan="2">France</td><td>River</td><td>Pseudomonas fluorescens (1)</td><td>-</td><td>-</td><td>[67]</td></tr><tr><td>River</td><td>Enterobacter kobei (1), Aeromonas (1), Acinetobacter (1)</td><td>-</td><td>-</td><td>[42]</td></tr><tr><td rowspan="3">GES-16</td><td rowspan="3">Brazil</td><td>River</td><td>Klebsiella pneumoniae (2)</td><td>ST1793, ST1794</td><td>-</td><td>-</td></tr><tr><td>Enterobacter (2), Klebsiella (1)</td><td>-</td><td>-</td><td>[25]</td></tr><tr><td>Enterobacter kobei (1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="2">GES-20</td><td rowspan="2">Philippines</td><td>River</td><td>Klebsiella oxytoca (1), Enterobacter kobei (1)</td><td>-</td><td>-</td><td>[64]</td></tr><tr><td>River</td><td>Klebsiella variicola (1)</td><td>ST2790</td><td>-</td><td>[53]</td></tr><tr><td>GES-24</td><td>Japan</td><td>WWTP</td><td>Aeromonas punctata (1)</td><td>-</td><td>-</td><td>[42]</td></tr><tr><td rowspan="3">GES-31</td><td rowspan="3">Brazil</td><td>River</td><td>Enterobacter asburiae (1)</td><td>-</td><td>-</td><td>[23]</td></tr><tr><td>River</td><td>Enterobacter cloacae (1)</td><td>ST822</td><td>IncFIB</td><td>[24]</td></tr><tr><td>River</td><td>Enterobacter asburiae (1)</td><td>-</td><td>-</td><td>[22]</td></tr><tr><td rowspan="3">IMI-2</td><td rowspan="3">France</td><td>River</td><td>Enterobacter asburiae (7)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>River</td><td>Enterobacter cloacae (1)</td><td>-</td><td>-</td><td>[64]</td></tr><tr><td>River</td><td>Pseudomonas aeruginosa (14)</td><td>-</td><td>-</td><td>[117]</td></tr><tr><td rowspan="3">KPC</td><td>Egypt</td><td>Drinking water</td><td>Klebsiella pneumoniae (5)</td><td>-</td><td>-</td><td>[27]</td></tr><tr><td>Ireland</td><td>Hospital sewage</td><td>Citrobacter freundii (4)</td><td>-</td><td>-</td><td>[32]</td></tr><tr><td>Jordan</td><td>Drinking water</td><td>Escherichia coli (5)</td><td>-</td><td>-</td><td>[34]</td></tr><tr><td rowspan="2">Korea</td><td>Korea</td><td>Water dispenser</td><td>Escherichia coli (1)</td><td>-</td><td>-</td><td>[35]</td></tr><tr><td>Singapore</td><td>Hospital sewage</td><td>Enterobacter cloacae (1), Enterobacter kobei (1), Enterobacter asburiae (1)</td><td>-</td><td>-</td><td>[74]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>USA</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (8), Citrobacter (1), Enterobacter (9), Pseudomonas (4) Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Aeromonas, Acinetobacter (total, 40)</td><td>-</td><td>IncN</td><td>[92]</td></tr><tr><td>Aeromonas (23), Serratia marcescens (70), Klebsiella pneumoniae (1), Klebsiella oxytoca (6), Enterobacter cloacae complex (15), Kluyvera intermedia (1), Pantoea (3), Citrobacter freundii (12), Raoultella (4), other Enterobacteriaceae (6) WWTP</td><td>KPC-2</td><td>Austria</td><td>Escherichia coli (21) Klebsiella pneumoniae (1) WWTP</td><td>-</td><td>[31]</td><td></td></tr><tr><td></td><td>KPC-2</td><td>Brazil</td><td>Escherichia coli (2) Klebsiella pneumoniae (2) Enterobacter (1), Enterobacter cloacae (1), Klebsiella pneumoniae (11) Klebsiella (25), Enterobacter (26), Serratia (3), Raoultella (4), Kluyvera (5) Pseudomonas putida (2), Stenotrophomonas maltophilia (4) Klebsiella pneumoniae (3) Aeromonas hydrophila (1), Aeromonas punctata (2) Klebsiella pneumoniae (7) Enterobacter cloacae (3), Enterobacter kobei (12) Enterobacter asburiae (6) Klebsiella pneumoniae (1), Enterobacter cloacae (1) Enterobacter (9), Citrobacter (1), Kluyvera (2) Aeromonas (1), Enterobacter (9), Citrobacter (1), Kluyvera (2)</td><td>ST1245</td><td>-</td><td>[30]</td><td>[39]</td></tr><tr><td></td><td></td><td>Brazil</td><td>Hospital sewage</td><td>-</td><td>-</td><td>[37]</td></tr><tr><td></td><td></td><td>Hospital sewage, WWTP</td><td>-</td><td>-</td><td>-</td><td>[47]</td></tr><tr><td></td><td></td><td>Mangroves</td><td>-</td><td>-</td><td>-</td><td>[40]</td></tr><tr><td></td><td></td><td>River</td><td>-</td><td>-</td><td>-</td><td>[68]</td></tr><tr><td></td><td></td><td>River</td><td>ST437, ST340</td><td>IncN</td><td>-</td><td>[41]</td></tr><tr><td></td><td></td><td>River</td><td>-</td><td>-</td><td>-</td><td>[42]</td></tr><tr><td></td><td></td><td>River</td><td>ST1792, ST1791, ST1245, ST1793, ST1794, ST1795</td><td>-</td><td>-</td><td>[47]</td></tr><tr><td></td><td></td><td>River</td><td>-</td><td>-</td><td>-</td><td>[47]</td></tr><tr><td></td><td></td><td>Seawater</td><td>-</td><td>-</td><td>-</td><td>[25]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td></td><td></td><td></td><td>Citrobacter (1), Enterobacter (65), Klebsiella (17), Kluyvera (2), Serratia (7), Aeromonas sp. (5)</td><td>-</td><td>-</td><td>[48]</td></tr><tr><td></td><td>Bulgaria</td><td>WWTP</td><td>Aeromonas (4)</td><td>-</td><td>-</td><td>[40]</td></tr><tr><td></td><td></td><td>River</td><td>Enterobacter asburiae (1)</td><td>-</td><td>-</td><td>[44]</td></tr><tr><td></td><td>China</td><td>Hospital sewage</td><td>Enterobacter cloacae (3), Citrobacter freundii (5)</td><td>-</td><td>IncA/C</td><td>[38]</td></tr><tr><td></td><td></td><td></td><td>Citrobacter freundii (1)</td><td>ST88</td><td>-</td><td>[85]</td></tr><tr><td></td><td></td><td></td><td>Citrobacter freundii (1)</td><td>ST14</td><td>-</td><td>[52]</td></tr><tr><td></td><td></td><td></td><td>Enterobacter cloacae (4)</td><td>ST911, ST910, ST25, ST669</td><td>-</td><td>[52]</td></tr><tr><td></td><td>China</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (4)</td><td>ST11, ST12</td><td>-</td><td>[52]</td></tr><tr><td></td><td></td><td></td><td>Klebsiella pneumoniae (3), Escherichia coli (1), Citrobacter sp. (1), Citrobacter braakii (1), Raoultella planticola (2), Enterobacter sp. (1), Enterobacter kobei (1)</td><td>-</td><td>-</td><td>[96]</td></tr><tr><td></td><td></td><td>River</td><td>Aeromonas hydrophila (3)</td><td>-</td><td>-</td><td>[49]</td></tr><tr><td></td><td></td><td></td><td>Citrobacter braakii (2), Citrobacter freundii (2)</td><td>-</td><td>IncF</td><td>[50]</td></tr><tr><td></td><td></td><td></td><td>Escherichia coli (4), Kluyvera georgiana (1)</td><td>-</td><td>-</td><td>[50]</td></tr><tr><td></td><td></td><td>Wells</td><td>Raoultella ornithinolytica (1)</td><td>-</td><td>-</td><td>[51]</td></tr><tr><td></td><td></td><td>WWTP</td><td>Klebsiella (10), Shigella (17), Escherichia (12)</td><td>-</td><td>IncF</td><td>[43]</td></tr><tr><td></td><td></td><td></td><td>Acinetobacter (19), Stenotrophomonas (10), Wautersiella (9)</td><td>-</td><td>-</td><td>[50]</td></tr><tr><td></td><td></td><td></td><td>Raoultella terrigena (1), Escherichia coli (8), Kluyvera georgiana (2), Acinetobacter seohaensis (3), Shigella sonnei (1)</td><td>-</td><td>-</td><td>[50]</td></tr><tr><td>Croatia</td><td>River</td><td></td><td>Klebsiella pneumoniae (4)</td><td>ST258</td><td>IncFII</td><td>[56]</td></tr><tr><td>Italy</td><td>WWTP</td><td></td><td>Klebsiella pneumoniae (1)</td><td>ST307</td><td>IncFIIK</td><td>[45]</td></tr><tr><td></td><td>Japan</td><td>WWTP</td><td>Klebsiella pneumoniae (1)</td><td>ST11</td><td>IncFII, IncN</td><td>[54]</td></tr><tr><td></td><td></td><td></td><td>Aeromonas caviae (1)</td><td>ST13</td><td>IncP6</td><td>[66]</td></tr><tr><td></td><td></td><td></td><td>Aeromonas hydrophila (1)</td><td>ST 558</td><td>IncP6</td><td>[66]</td></tr><tr><td>Philippines</td><td>Hospital sewage</td><td></td><td>Citrobacter freundii (1), Klebsiella pneumoniae (2)</td><td>-</td><td>-</td><td>[64]</td></tr><tr><td></td><td>River</td><td></td><td>Escherichia coli (2), Klebsiella pneumoniae (2)</td><td>-</td><td>-</td><td>[64]</td></tr><tr><td>Portugal</td><td>River</td><td></td><td>Escherichia coli (1)</td><td>ST410</td><td>IncF</td><td>[36]</td></tr><tr><td>Romania</td><td>Hospital sewage</td><td></td><td>Klebsiella pneumoniae (9)</td><td>-</td><td>-</td><td>[97]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">Spain</td><td rowspan="10">River</td><td>River</td><td>Klebsiella pneumoniae (1)<br>Enterobacter cloacae (1)<br>Klebsiella pneumoniae (1)<br>Klebsiella oxytoca (1)<br>Escherichia coli (3)</td><td>ST823<br>ST634<br>ST1434, ST5001, ST216</td><td>IncN, IncR, IncFIIK</td><td>[24]</td></tr><tr><td>WWTP</td><td>Citrobacter freundii (5), Citrobacter braakii (1), Citrobacter farmeri (1), Enterobacter cloacae/asburiae (5), Klebsiella oxytoca (6), Klebsiella pneumoniae (1), Kluyvera ascorbata (1), Kluyvera cryocrescens (2), Raoultella ornithinolytica (3)</td><td>ST1434, ST5001, ST216</td><td>IncN, IncR, IncFIIK</td><td>[55]</td></tr><tr><td>WWTP</td><td>Klebsiella pneumoniae (1)</td><td>ST258</td><td>IncP/6</td><td>[46]</td></tr><tr><td>Hospital sewage</td><td>Klebsiella pneumoniae (4)</td><td>ST2256, ST512, ST258</td><td>IncP6</td><td>[53]</td></tr><tr><td>Taiwan</td><td>Enterobacter kobei (1)<br>Klebsiella quasipneumoniae (1)</td><td>ST910<br>ST2786</td><td>IncP6</td><td>[53]</td></tr><tr><td>USA</td><td>Enterobacter cloacae (2)<br>Enterobacter cloacae complex (2)<br>Klebsiella pneumoniae (3)<br>Klebsiella quasipneumoniae (1)<br>Klebsiella oxytoca (2)<br>Aeromonas caviae (2)<br>Raoultella ornithinolytica (1)<br>Aeromonas caviae (2)<br>Enterobacter cloacae complex (3)<br>Enterobacter cloacae (1)<br>Enterobacter asburiae (1)<br>Citrobacter freundii (1)</td><td>ST1121, ST1122<br>ST595, ST1028<br>ST3539, ST872, ST2793<br>ST138<br>ST88, ST127<br>ST560, ST561, ST563<br>ST564, ST562<br>ST131, ST928, ST595<br>ST41<br>ST24<br>ST8</td><td>IncP6</td><td>[57]</td></tr><tr><td>KPC-3</td><td>Italy</td><td>Wells</td><td>Klebsiella pneumoniae (1)<br>Klebsiella pneumoniae (20)</td><td>ST258<br>ST512</td><td>IncFIIK</td><td>[45]</td></tr><tr><td>Portugal</td><td>River</td><td>Klebsiella pneumoniae (9)<br>Enterobacter cloacae (2)</td><td>ST512</td><td>IncFIIK</td><td>[58]</td></tr><tr><td>USA</td><td>WWTP</td><td>Citrobacter freundii (2)<br>Escherichia coli (1)<br>Enterobacter (2), Klebsiella (1)<br>Serratia fonticola (1)</td><td>ST413, ST11<br>ST607<br>ST516</td><td>IncFIIK</td><td>[59]</td></tr><tr><td>KPC-26</td><td>Brazil</td><td>Seawater</td><td>Escherichia coli (1)</td><td>ST607</td><td>IncFIIK</td><td>[25]</td></tr><tr><td>SFC-1</td><td>Portugal</td><td>Drinking water</td><td>Serratia fonticola (1)</td><td>ST607</td><td>IncFIIK</td><td>[20]</td></tr><tr><td>VCC-1</td><td>Germany</td><td>Seawater</td><td>Vibrio cholerae (3)</td><td>ST516</td><td>IncFIIK</td><td>[69]</td></tr></tbody></table>

n, number of strains, ST, sequence type; WWTP, wastewater treatment plant.
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Worldwide distribution of carbapenemase-producing Gram-negative bacteria in aquatic environments.</figcaption></figure>

Brazilian river [42]. de Araujo et al. and Xu et al. have reported the isolation of KPC-2-producing *Aeromonas hydrophila* from rivers in Brazil and China, respectively [42,49]. Recently, KPC-2-producing *A. hydrophilia* and *Aeromonas caviae* were recovered from river water and WWTP effluents [57,66].

Although rarely identified, Haller et al. have reported the isolation of KPC-producing *Pseudomonas* spp. from hospital sewage in Singapore [28]. Furthermore, BIC-1 (GES-7)-producing *Pseudomonas fluorescens* was obtained from a river in France [67]. More recently, Neto et al. have detected KPC-2-producing *Pseudomonas putida* in mangroves in Brazil [68].

In addition, KPC-2-producing *Stenotrophomonas* spp. and *Wautersiella* spp. isolates were recovered from a WWTP in China [43] and KPC-2-producing *Stenotrophomonas maltophilia* was recovered from mangroves in Brazil [68]. Finally, Hammerl et al. have documented the isolation of VCC-1 (for *Vibrio cholerae* carbapenemase)-producing *V. cholerae* from seawater in Germany [69].

## 3.2. Class B carbapenemases

Unlike class A carbapenemases, which were first reported in Enterobacterales, the first acquired class B β-lactamase (BCII) was detected in a *Bacillus cereus* isolate in 1966 [70]. Owing to the fact that they use one or two zinc (Zn²⁺) ions for their activity, these enzymes are also called metallo-β-lactamases (MBLs)

[71] and this property makes them susceptible to inhibition by metallic ion chelators such as ethylene diamine tetra-acetic acid (EDTA) [72]. However, they are resistant to the commercially available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam) [42]. MBLs breakdown all β-lactams except monobactams (aztreonam), with VIM, IMP and NDM groups being the most commonly identified acquired MBLs [15]. The global epidemiology of class B carbapenemases in aquatic environments is presented in Table 2 and Fig. 3.

### 3.2.1. Enterobacterales

NDM enzymes are among the most newly characterised MBLs, which are widely distributed in Enterobacterales species [73]. The *bla*~NDM~ genes have been detected in *E. cloacae*, *E. coli*, *C. freundii*, *C. braakii*, *C. farmeri*, *K. pneumoniae*, *K. oxytoca* and *Shigella boydii* isolated from municipal and hospital sewage [32,33,61,74–76]. Mahon et al. have reported the isolation of NDM-producing *E. coli* from recreational freshwater and wastewater [77]. Furthermore, NDM-producing *K. pneumoniae* and *K. quasipneumoniae* could also be isolated from sewage and seawater [76,77]. More worryingly, two studies have reported the isolation of NDM-producing *K. pneumoniae* and *E. coli* from drinking water in Egypt and Jordan, respectively [27,34]. Several NDM variants have been detected in Enterobacterales species isolated from aqueous ecosys-
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Phylogenetic tree of class A carbapenemase variants detected in different aquatic environments. The evolutionary history was inferred using the neighbour-joining method. Evolutionary distances were computed using the Kimura 2-parameter method. The number above the nodes is the level of bootstrap from 1000 replicates.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Phylogenetic tree of class B carbapenemase variants detected in different aquatic environments. The evolutionary history was inferred using the neighbour-joining method. Evolutionary distances were computed using the Kimura 2-parameter method. The number above the nodes is the level of bootstrap from 1000 replicates.</figcaption></figure>
<table><caption>**Table 2**
Epidemiology of class B carbapenemase-producers detected in aquatic environments</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td>GIM</td><td>Germany</td><td>Clinical and urban WW</td><td>Enterobacter cloacae complex (1)</td><td>–</td><td>–</td><td>[76]</td></tr><tr><td>IMP</td><td>Ireland</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (2)<br>Enterobacter cloacae complex (3)</td><td>ST3146</td><td>–</td><td>[32]</td></tr><tr><td></td><td>Singapore</td><td>Hospital sewage</td><td>Aeromonas caviae (1)</td><td>–</td><td>–</td><td>[74]</td></tr><tr><td rowspan="3">IMP-1</td><td>USA</td><td>WWTP</td><td>Escherichia coli (1)</td><td>–</td><td>–</td><td>[30]</td></tr><tr><td>Brazil</td><td>Seawater</td><td>Acinetobacter (3)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>UK</td><td>WWTP</td><td>Enterobacter cloacae (1)</td><td>–</td><td>–</td><td>[63]</td></tr><tr><td>IMP-4</td><td>Australia</td><td>Hospital sink water</td><td>Serratia marcescens (4)</td><td>–</td><td>–</td><td>[104]</td></tr><tr><td rowspan="2"></td><td>China</td><td>Incubator water</td><td>Klebsiella pneumoniae (1)</td><td>ST105</td><td>IncFI</td><td>[83]</td></tr><tr><td></td><td>Hospital sewage</td><td>Citrobacter freundii (1)</td><td>–</td><td>–</td><td>[38]</td></tr><tr><td>IMP-7</td><td>Brazil</td><td>Seawater</td><td>Acinetobacter (1)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td rowspan="2">IMP-8</td><td>Portugal</td><td>River</td><td>Escherichia coli (2)</td><td>ST2612</td><td>IncFIB</td><td>[106]</td></tr><tr><td>Spain</td><td>Hospital WW drainage system</td><td>Klebsiella oxytoca (1)</td><td>–</td><td>–</td><td>[105]</td></tr><tr><td></td><td>Taiwan</td><td>Hospital sewage</td><td>Enterobacter hormaechei (2)<br>Citrobacter (1)</td><td>ST90</td><td>–</td><td>[53]</td></tr><tr><td>IMP-16</td><td>Brazil</td><td>Seawater</td><td>Acinetobacter (4)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>IMP-19</td><td>Taiwan</td><td>Hospital sewage</td><td>Klebsiella quasipneumoniae (1)</td><td>ST2789</td><td>–</td><td>[53]</td></tr><tr><td>IMP-25</td><td>China</td><td>Hospital sewage</td><td>Pseudomonas (1)</td><td>–</td><td>–</td><td>[116]</td></tr><tr><td>IMP-29</td><td>Sweden</td><td>WWTP</td><td>Klebsiella oxytoca (1)</td><td>ST192</td><td>–</td><td>[107]</td></tr><tr><td>KHM-1</td><td>Japan</td><td>WWTP</td><td>Klebsiella quasipneumoniae (1)</td><td>–</td><td>IncA/C2</td><td>[111]</td></tr><tr><td rowspan="2">NDM</td><td>Egypt</td><td>DW</td><td>Klebsiella pneumoniae (6)</td><td>–</td><td>–</td><td>[27]</td></tr><tr><td>Germany</td><td>Clinical WW</td><td>Enterobacter cloacae complex (2), Klebsiella oxytoca (1)</td><td>–</td><td>–</td><td>[76]</td></tr><tr><td></td><td>Clinical and urban WW</td><td>Klebsiella quasivariicola (1), Klebsiella pneumoniae (3)</td><td>–</td><td>–</td><td>–</td><td></td></tr><tr><td rowspan="3">Ireland</td><td>Hospital sewage</td><td>Escherichia coli (1)</td><td>ST617</td><td>–</td><td>–</td><td>[32]</td></tr><tr><td>FSW, WW</td><td>Escherichia coli (5)</td><td>–</td><td>–</td><td>–</td><td>[77]</td></tr><tr><td>Seawater</td><td>Klebsiella pneumoniae (11)</td><td>–</td><td>–</td><td>–</td><td></td></tr><tr><td rowspan="2">Jordan</td><td>DW</td><td>Escherichia coli (12)</td><td>–</td><td>–</td><td>–</td><td>[34]</td></tr><tr><td>Singapore</td><td>Hospital sewage</td><td>Enterobacter cloacae (1), Escherichia coli (1), Citrobacter freundii (1)</td><td>–</td><td>–</td><td>[74]</td></tr><tr><td>UK</td><td>Hospital sewage</td><td>Enterobacter cloacae complex (2), Citrobacter freundii (4)</td><td>–</td><td>–</td><td>–</td><td>[61]</td></tr><tr><td>NDM-1</td><td>Bangladesh</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (46), Escherichia coli (30), Citrobacter freundii (2), Providencia rettgeri (1), Enterobacter (9), Acinetobacter baumannii (8), Acinetobacter pittii (4), Acinetobacter baylyi (1), Acinetobacter spp. (3)</td><td>–</td><td>–</td><td>[89]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>Bangladesh</td><td>Wastewater</td><td>Escherichia coli (49)<br>Klebsiella pneumoniae (76), Citrobacter freundii (10)<br>Pseudomonas spp. (2), Pseudomonas aeruginosa (4), Pseudomonas putida (24), Pseudomonas otitidis (10), Pseudomonas monteilii (3), Pseudomonas mendocina (8), Pseudomonas fulva (3), Pseudomonas oleovorans (7), Pseudomonas alcaligenes (1), Pseudomonas pseudoalcaligenes (3), Aeromonas caviae (1), Acinetobacter baumannii (2), Acinetobacter tandoii (4), Acinetobacter towneri (5), unidentified (15)</td><td>ST101, ST648, ST405<br>-</td><td>IncX, IncFII<br>-</td><td>[81]</td></tr><tr><td>Brazil</td><td>Seawater</td><td></td><td>Klebsiella pneumoniae (1)<br>Klebsiella (1)</td><td>ST11<br>-</td><td>IncA/C<br>-</td><td>[84]</td></tr><tr><td>China</td><td>Animal waste</td><td></td><td>Escherichia coli (1)<br>Citrobacter freundii (2)<br>Cronobacter sakazakii (1)<br>Klebsiella pneumoniae (1)<br>Providencia rettgeri (1)<br>Enterobacter cloacae (1)</td><td>ST416<br>-<br>ST3410<br>-<br>ST591</td><td>IncX3<br>IncA/C<br>-<br>IncA/C<br>-</td><td>[48]<br>[93]<br>[98]</td></tr><tr><td></td><td>Hospital sewage</td><td></td><td>Acinetobacter johnsonii (2)<br>Acinetobacter baumannii (10)<br>Citrobacter freundii (1)<br>Enterobacter cloacae (2)<br>Citrobacter freundii (2)<br>Acinetobacter chinensis (2)<br>Kluyvera cryocrescens (1)<br>Raoultella planticola (1), Escherichia coli (3), Citrobacter sp. (1), Acinetobacter (11)</td><td>ST88<br>ST910<br>ST19, ST22<br>-<br>-<br>-<br>-</td><td>IncX4<br>IncX3<br>-<br>-<br>-<br>-</td><td>[113]<br>[114]<br>[85]<br>[52]</td></tr><tr><td></td><td>Incubator water</td><td></td><td>Klebsiella pneumoniae (1)<br>Acinetobacter seohaensis (2), Stenotrophomonas maltophilia (1)<br>Escherichia coli (1), Acinetobacter (1)</td><td>ST105<br>-</td><td>IncFI<br>IncA/C<br>-</td><td>[83]<br>[86]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">India</td><td rowspan="10">Hospital sewage</td><td>WWTP</td><td>Achromobacter sp. (1)<br>Escherichia coli (3), Shigella sonnei (2), Acinetobacter seohaensis (4), Wautersiella falsenii (1)</td><td>-</td><td>-</td><td>[122]</td></tr><tr><td>Escherichia coli (1)</td><td>-</td><td>IncA/C</td><td>[43]</td></tr><tr><td>Escherichia coli (1), Citrobacter freundii (7)</td><td>-</td><td>-</td><td>[80]</td></tr><tr><td>Acinetobacter baumannii (11), Acinetobacter calcoaceticus (1), Acinetobacter junii (1), Aeromonas hydrophila (2)</td><td>-</td><td>IncFIC, IncH11, IncFIA, IncP, IncI1</td><td>[75]</td></tr><tr><td>Aeromonas veronii (2), Cronobacter malonaticus (1), Enterobacter cancerogenus (1), Enterobacter cloacae (1), Enterobacter kobei (1), Enterobacter ludwigii (1), Escherichia coli (14), Klebsiella pneumoniae (10), Klebsiella aerogenes (1)</td><td>-</td><td>-</td><td>[95]</td></tr><tr><td>River, lake, DW</td><td>Klebsiella pneumoniae (3), Shigella (2), Escherichia coli (1), Citrobacter freundii (1), Pseudomonas (3)</td><td>-</td><td>-</td><td>[79]</td></tr><tr><td>Seawater</td><td>Pseudomonas putida (2), Pseudomonas pseudoalcaligenes (2), Pseudomonas oryzihabitans (1), Suttonella indologenes (1), Aeromonas caviae (1), Stenotrophomonas maltophilia (1), Vibrio cholerae (1)</td><td>-</td><td>IncA/C</td><td>[78]</td></tr><tr><td>Tap water</td><td>Achromobacter spp. (2), Kingella denitrificans (1), Pseudomonas aeruginosa (1)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Lebanon</td><td>WWTP</td><td>Escherichia coli (2)</td><td>-</td><td>IncA/C, IncFIIK</td><td>[87]</td></tr><tr><td>Myanmar</td><td>Hospital sewage</td><td>Enterobacter cloacae (2)</td><td>-</td><td>-</td><td>[90]</td></tr><tr><td>Nigeria</td><td>Hospital sewage</td><td>Citrobacter freundii (1)</td><td>-</td><td>IncX3</td><td>[91]</td></tr><tr><td></td><td></td><td>Klebsiella pneumoniae (1)</td><td>ST147</td><td>IncX3</td><td></td></tr><tr><td></td><td>Seawater</td><td>Vibrio parahaemolyticus (1), Vibrio vulnificus (5)</td><td>-</td><td>-</td><td>[123]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>Philippines</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (4), Klebsiella oxytoca (1), Enterobacter cloacae (4), Enterobacter xiangfangensis (1), Citrobacter amalonaticus (1), Serratia marcescens (1), Kluyvera ascorbata (1)</td><td>-</td><td>-</td><td>[64]</td></tr><tr><td></td><td>River</td><td>River</td><td>Escherichia coli (1), Klebsiella pneumoniae (1), Enterobacter cloacae (1), Enterobacter tabaci (1), Enterobacter xiangfangensis (1), Enterobacter hormaechei (1), Citrobacter freundii (1)</td><td>-</td><td>-</td><td></td></tr><tr><td></td><td>Portugal</td><td>River</td><td>Enterobacter spp. (3)</td><td>-</td><td>IncA/C2</td><td>[59]</td></tr><tr><td></td><td>Romania</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (7)</td><td>-</td><td>-</td><td>[97]</td></tr><tr><td></td><td>River</td><td>River</td><td>Klebsiella pneumoniae (1)</td><td>-</td><td>-</td><td>[44]</td></tr><tr><td></td><td>Saudi Arabia</td><td>WWTP</td><td>Escherichia coli (1)</td><td>ST101</td><td>IncF</td><td>[82]</td></tr><tr><td></td><td>Singapore</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (3), Enterobacter (3), Citrobacter (6), Pseudomonas (9), Acinetobacter (4), Aeromonas hydrophila (1)</td><td>-</td><td>-</td><td>[28]</td></tr><tr><td></td><td>South Korea</td><td>Hospital sinks</td><td>Enterobacter cloacae (1), Citrobacter freundii (1)</td><td>-</td><td>-</td><td>[35]</td></tr><tr><td></td><td>Switzerland</td><td>Hospital sewage</td><td>Citrobacter sp. (1)</td><td>-</td><td>-</td><td>[46]</td></tr><tr><td></td><td>WWTP</td><td>WWTP</td><td>Citrobacter sp. (1)</td><td>-</td><td>-</td><td></td></tr><tr><td></td><td>Taiwan</td><td>Hospital sewage</td><td>Klebsiella pneumoniae (1)</td><td>ST11</td><td>-</td><td>[53]</td></tr><tr><td></td><td>USA</td><td>River</td><td>Escherichia coli (1)</td><td>ST410</td><td>-</td><td>[57]</td></tr><tr><td></td><td>Vietnam</td><td>River</td><td>Klebsiella pneumoniae (3)</td><td>-</td><td>-</td><td>[99]</td></tr><tr><td></td><td>UK</td><td>WWTP</td><td>Klebsiella pneumoniae (1)</td><td>-</td><td>-</td><td>[63]</td></tr><tr><td>NDM-3</td><td>Bangladesh</td><td>Wastewater</td><td>Escherichia coli (3)</td><td>ST101</td><td>IncFII</td><td>[81]</td></tr><tr><td>NDM-4</td><td>India</td><td>Hospital sewage</td><td>Escherichia coli (1)</td><td>ST648</td><td>IncX, IncFII</td><td></td></tr><tr><td></td><td></td><td></td><td>Escherichia coli (1)</td><td>-</td><td>IncK</td><td>[100]</td></tr><tr><td></td><td></td><td></td><td>Escherichia coli (8), Citrobacter braakii (2), Citrobacter farmeri (1)</td><td>-</td><td>IncFIA, IncP, IncY, IncFIB, IncFIC, IncH11, IncI1</td><td>[75]</td></tr><tr><td></td><td>Myanmar</td><td>Hospital sewage</td><td>Enterobacter asburiae (1), Leclercia adecarboxylata (1), Citrobacter freundii (2), Klebsiella quasipneumoniae (1)</td><td>-</td><td>IncX3</td><td>[91]</td></tr><tr><td>NDM-5</td><td>China</td><td>Animal waste</td><td>Klebsiella pneumoniae (3)</td><td>ST37, ST726</td><td>IncX3</td><td>[98]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="12">NDM-7</td><td rowspan="12">China</td><td rowspan="4">Hospital sewage</td><td>Klebsiella oxytoca (1)</td><td>ST158</td><td rowspan="12">IncX3</td><td rowspan="12">[101]</td></tr><tr><td>Enterobacter cloacae (4)</td><td>ST1</td></tr><tr><td>Cronobacter sakazakii (1)</td><td>–</td></tr><tr><td>Citrobacter freundii (9)</td><td>ST109</td></tr><tr><td rowspan="4">Hospital sewage</td><td>Escherichia coli (6)</td><td>ST6751, ST746</td></tr><tr><td>Escherichia coli (1)</td><td>ST410</td></tr><tr><td>Klebsiella pneumoniae (3), Escherichia coli (9), Acinetobacter (1), Klebsiella variicola (1)</td><td>–</td></tr><tr><td>River</td><td>–</td></tr><tr><td rowspan="4">Hospital sewage</td><td>Klebsiella pneumoniae (1), Escherichia coli (1), Citrobacter freundii (1)</td><td>–</td></tr><tr><td>Escherichia coli (6), Shigella boydii (4)</td><td>–</td></tr><tr><td>WWTP, rivers</td><td>–</td></tr><tr><td>WWTP</td><td>–</td></tr><tr><td rowspan="4">Hospital sewage</td><td>Escherichia coli (7)</td><td>ST542</td></tr><tr><td>Escherichia coli (1)</td><td>–</td></tr><tr><td>Klebsiella pneumoniae (1)</td><td>ST8453</td></tr><tr><td>Escherichia coli (4)</td><td>–</td></tr><tr><td rowspan="3">Hospital sewage</td><td>Escherichia coli (1)</td><td>ST617</td></tr><tr><td>Escherichia coli (1)</td><td>ST10, ST216, ST2914</td></tr><tr><td>Escherichia coli (1)</td><td>–</td></tr><tr><td rowspan="3">Hospital sewage</td><td>Escherichia coli (1)</td><td>ST167</td></tr><tr><td>Escherichia coli (1)</td><td>–</td></tr><tr><td>Escherichia coli (1), Citrobacter freundii (1)</td><td>–</td></tr><tr><td rowspan="3">Hospital sewage</td><td>Escherichia coli (5)</td><td>–</td></tr><tr><td>Escherichia coli (1)</td><td>–</td></tr><tr><td>Escherichia coli (2), Klebsiella pneumoniae (1), Enterobacter cloacae (3), Citrobacter freundii (4)</td><td>–</td></tr><tr><td rowspan="4">Hospital sewage</td><td>Escherichia coli (3), Klebsiella pneumoniae (1), Klebsiella oxytoca (1), Citrobacter amalonaticus (1), Raoultella ornithinolytica (1)</td><td>–</td></tr><tr><td>Klebsiella pneumoniae (1)</td><td>ST19</td></tr><tr><td>Klebsiella variicola (3)</td><td>–</td></tr><tr><td>Klebsiella pneumoniae (1)</td><td>ST147</td></tr><tr><td rowspan="2">Hospital sewage</td><td>Serratia fonticola (1)</td><td>–</td></tr><tr><td>Pseudomonas aeruginosa (6)</td><td>–</td></tr><tr><td rowspan="2">Hospital sewage</td><td>Pseudomonas aeruginosa (1)</td><td>–</td></tr><tr><td>Pseudomonas aeruginosa (14)</td><td>–</td></tr><tr><td rowspan="2">Hospital sewage</td><td>Enterobacter cloacae complex (1), Enterobacter (1), Pseudomonas aeruginosa (8)</td><td>–</td></tr><tr><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="12">VIM-1</td><td>Germany</td><td>WWTP</td><td>Pseudomonas aeruginosa (1), Escherichia coli (3)</td><td>–</td><td>–</td><td>[108]</td></tr><tr><td>Ireland</td><td>Hospital sewage</td><td>Klebsiella oxytoca (1)</td><td>ST202</td><td>–</td><td>[32]</td></tr><tr><td></td><td></td><td>Enterobacter cloacae complex (1)</td><td>–</td><td>–</td><td></td></tr><tr><td>Jordan</td><td>DW</td><td>Escherichia coli (75)</td><td>–</td><td>–</td><td>[34]</td></tr><tr><td>Switzerland</td><td>WWTP</td><td>Enterobacter aerogenes (1)</td><td>–</td><td>–</td><td>[46]</td></tr><tr><td>USA</td><td>WWTP</td><td>Escherichia coli (36)</td><td>–</td><td>–</td><td>[30]</td></tr><tr><td>Austria</td><td>River</td><td>Enterobacter cloacae (1)</td><td>–</td><td>IncHI2</td><td>[88]</td></tr><tr><td>Portugal</td><td>River</td><td>Escherichia coli (1)</td><td>ST167</td><td>IncFIB</td><td>[106]</td></tr><tr><td>Spain</td><td>River</td><td>Raoultella ornithinolytica (1)</td><td>–</td><td>IncR</td><td>[24]</td></tr><tr><td>Sweden</td><td>River</td><td>Klebsiella oxytoca (1)</td><td>ST172</td><td>–</td><td>[107]</td></tr><tr><td>Switzerland</td><td>Hospital sewage</td><td>Citrobacter (1), Citrobacter freundii (1)</td><td>–</td><td>–</td><td>[46]</td></tr><tr><td></td><td></td><td>Escherichia coli (1)</td><td>ST393</td><td>–</td><td></td></tr><tr><td></td><td>River</td><td>Klebsiella pneumoniae (1)</td><td>–</td><td>–</td><td>[109]</td></tr><tr><td>Taiwan</td><td>Hospital sewage</td><td>Klebsiella quasipneumoniae (1)</td><td>ST2788</td><td>–</td><td>[53]</td></tr><tr><td rowspan="7">VIM-2</td><td>USA</td><td>River</td><td>Raoultella planticola (1)</td><td>–</td><td>–</td><td>[57]</td></tr><tr><td>Brazil</td><td>Seawater</td><td>Pseudomonas (2)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>China</td><td>Hospital sewage</td><td>Pseudomonas (1)</td><td>–</td><td>–</td><td>[116]</td></tr><tr><td>Portugal</td><td>River</td><td>Pseudomonas putida (1), Pseudomonas geniculata (1), Pseudomonas monteilii (1)</td><td>–</td><td>–</td><td>[119]</td></tr><tr><td></td><td></td><td>Pseudomonas sp. (2)</td><td>–</td><td>–</td><td></td></tr><tr><td>Wastewater</td><td></td><td>Pseudomonas pseudoalcaligenes (1)</td><td>–</td><td>–</td><td>[118]</td></tr><tr><td>Serbia</td><td>River</td><td>Acinetobacter baumannii (2)</td><td>–</td><td>–</td><td>[120]</td></tr><tr><td rowspan="2">VIM-5</td><td>Nigeria</td><td>Wetlands</td><td>Pseudomonas plecoglossicida (6), Pseudomonas guariconensis (4)</td><td>–</td><td>–</td><td>[121]</td></tr><tr><td></td><td></td><td>Escherichia coli (1)</td><td>ST354</td><td>–</td><td>[106]</td></tr><tr><td>VIM-34</td><td>Portugal</td><td>River</td><td></td><td></td><td></td><td></td></tr></tbody></table>

n, number of strains; ST, sequence type; WW, wastewater; WWTP, wastewater treatment plant; DW, drinking water; FSW, fresh surface water.

tems. NDM-1-producing Enterobacter spp., Klebsiella spp., Citrobacter spp., Shigella spp., E. coli, C. freundii, Citrobacter amalonaticus, Cronobacter malonaticus, Cronobacter sakazakii, S. boydii, K. pneumoniae, K. oxytoca, Klebsiella aerogenes, Kluyvera cryocrescens, K. ascarbata, Shigella sonnei, Providencia rettgeri, Enterobacter xiangfangensis, E. cloacae, Enterobacter tabaci, Enterobacter hormaechei, Enterobacter cancerogenus, Enterobacter ludwigii, S. marcescens and R. planticola have been isolated from seawater, drinking water, rivers, lakes, hospital sewage, wastewater, WWTPs, irrigation water and animal sewage [28,35,44,46,48,52,53,57,59,63,64,75,78–99]. Only one study reported the isolation of an NDM-3-producer from an aquatic environment. The isolate was obtained from wastewater and was identified as E. coli sequence type 101 (ST101) [81]. In addition, an NDM-4 variant was detected in E. coli, K. quasipneumoniae, C. freundii, C. braakii, C. farmeri, Leclercia adecarboxylata and E. asburiae isolates obtained from hospital sewage and wastewater [75,81,91,100]. The NDM-5 enzyme was detected in E. coli, K. pneumoniae, K. variicola, K. oxytoca, C. freundii, C. sakazakii, E. cloacae and S. boydii isolates obtained from WWTPs, rivers, animal

waste and hospital sewage [28,46,57,63,64,87,91,96,98,101,102]. In addition, NDM-7-producers, namely E. coli, C. freundii, C. amalonaticus, E. cloacae, K. pneumoniae, K. oxytoca and R. ornithinolytica, have been recovered from WWTPs, rivers and hospital sewage [57,64,75,87,91]. Finally, NDM-9-producing E. coli and K. variicola have been isolated from WWTPs and river water, respectively [46,103].

Other important MBLs in GNB are the IMP enzymes. IMP-producing E. coli, E. cloacae complex and K. pneumoniae isolates have been recovered from US WWTPs and from Irish hospital sewage [30,32]. In addition, IMP-1-producing E. cloacae has been detected in WWTPs in the UK [63]. IMP-4-producing C. freundii and S. marcescens were isolated from hospital wastewater [38,104]. In China, an IMP-4-producing K. pneumoniae strain co-producing NDM-1 was isolated from incubator water in a neonatal intensive care unit (NICU). This strain was clonally related to NDM-1- and IMP-4-producing K. pneumoniae recovered from an outbreak in the same NICU, and the authors suggested that the incubator water may be a reservoir for the diffusion of such MBL-producing GNB
[83]. Furthermore, IMP-8-producing *K. oxytoca*, *Citrobacter* spp. and *E. hormaechei* and IMP-19-producing *K. quasipneumoniae* have been isolated from hospital wastewater [53,105]. In addition, Kieffer et al. have reported the isolation of IMP-8-producing *E. coli* from river water [106]. Recently, IMP-29-producing *K. oxytoca* ST192 was isolated from sewage of a WWTP in Sweden [107].

Further studies reported the detection of other MBLs among Enterobacteriales isolated from water habitats. VIM-producing *E. coli*, *K. pneumoniae*, *K. oxytoca*, *Enterobacter aerogenes*, *E. cloacae* complex and *Enterobacter* spp. have been detected in drinking water, WWTPs and hospital sewage [30,32–34,46,76,108]. In addition, *bla*<sub>VIM-1</sub>-harbouring *K. pneumoniae*, *K. variicola*, *E. coli*, *E. cloacae*, *R. planticola* and *R. ornithinolytica* and VIM-34-producing *E. coli* have been recovered from rivers [24,57,88,106,109]. Furthermore, VIM-1-producing *Citrobacter* spp., *E. coli*, *C. freundii* and *K. quasipneumoniae* have been isolated from hospital sewage [46,53]. In addition, a VIM-1-producing *K. oxytoca* ST172 isolate was recovered from a river in Sweden [107].

Other less common carbapenemases were also detected in water environments. This is the case with Sfh-1-producing *S. fonti-cola* isolated from drinking water in Portugal [110] and with GIM-producing *E. cloacae* complex (for Germany IMipenemase) isolated from urban and clinical wastewater in Germany [76]. In addition, the KHM-1 (Kyorin Health Science MBL-1) enzyme first described in a clinical isolate in Korea was detected in a *K. quasipneumoniae* isolated from a WWTP in Japan [111,112].

### 3.2.2. Other Gram-negative bacilli

Several studies have reported the detection of MBL-producing *Acinetobacter* and *Pseudomonas* species in environmental water habitats. NDM-1-producing *Acinetobacter* spp., *A. baumannii*, *Acinetobacter calcoaceticus*, *Acinetobacter junii*, *Acinetobacter johnsonii* and *Pseudomonas* spp. have been recovered from hospital sewage in Bangladesh, China, Singapore and the Philippines [28,64,89,96,113,114]. The occurrence of such superbugs in WWTPs was also described in some studies. NDM-1-producing *Pseudomonas* spp., *Acinetobacter* spp. and *Acinetobacter seohaensis* were detected in WWTPs in Bangladesh and China [81,86]. NDM-producing *Pseudomonas* and *Acinetobacter* species were also described in surface water. NDM-producing *Pseudomonas* spp. were isolated from rivers and lakes in India in 2013 [79]. In addition, NDM-1 producing *Acinetobacter* spp. and *A. seohaensis* were isolated from river water in China [86, 96] and NDM-1-producing *P. putida*, *Pseudomonas pseudoalcaligenes* and *Pseudomonas oryzihabitans* were obtained from seawater in India [78]. Recently, Hu et al. have reported the description of NDM-1-producing *Acinetobacter chinensis*, a novel *Acinetobacter* species isolated from hospital sewage in China [115]. In addition, the NDM-5 variant was detected in *Acinetobacter* sp. isolated from hospital sewage in China [96]. Regarding IMP carbapenemases, four variants were detected in *Acinetobacter* and *Pseudomonas* species isolated from water. IMP-1, IMP-7 and IMP-16 variants were detected in *Acinetobacter* spp. isolates obtained from seawater in Brazil [48], and the IMP-25 variant was detected in *Pseudomonas* sp. recovered from hospital sewage in China [116]. VIM enzymes were also detected. Miranda et al. have reported the detection of VIM-producing *Pseudomonas* sp. in hospital sewage [117]. The VIM-2 carbapenemase was also detected in *Pseudomonas* sp., *P. pseudoalcaligenes* and *P. aeruginosa* from hospital sewage [76,116,118], in *Pseudomonas* spp., *P. putida*, *Pseudomonas monteili* and *Pseudomonas geniculata* from river water [119], in *Pseudomonas* spp. from seawater [48] and in *A. baumannii* in river water [120]. Recently, VIM-5-producing *P. putida* group, namely *Pseudomonas plecoglossicida* and *Pseudomonas guariconensis*, were obtained from wetlands in Nigeria [121]. In addition, SPM (Sao Paulo metallo-β-lactamase)-producing *P. aeruginosa* and

SPM-1-producing *Pseudomonas* spp. were recovered from hospital sewage and seawater, respectively [48,117].

On the other hand, carbapenemase-producers belonging to several GNB genera, other than *Pseudomonas* and *Acinetobacter*, could also be detected in different aquatic habitats. NDM-1-producing *A. hydrophila* and *A. caviae* were recovered from hospital sewage in Singapore and the Philippines and from seawater in India [28,64,78]. In addition, *bla*<sub>IMP</sub>-harbouring *A. caviae* was isolated from hospital wastewater in India [78]. Luo et al. have reported the isolation of NDM-1-producing *Achromobacter* sp. from a WWTP in China [122]. Isolates of *V. cholerae*, *Vibrio parahaemolyticus* and *Vibrio vulnificus* isolated from seawater in India and Nigeria were found to be NDM-1-producers [78,123]. NDM-1-producing *S. maltophilia* isolates were obtained from river and seawater in China and India, respectively [78,86]. Finally, NDM-1-producing *Suttonella indologenes* and *Wautersiella falsenii* were isolated from seawater and a WWTP, respectively [78,86].

### 3.3. Class D carbapenemases

Only some variants of the class D β-lactamases possess carbapenemase activity, the so-called carbapenem-hydrolysing class D β-lactamases (CHDLs) [19]. The first identified class D carbapenemase was the OXA-23 enzyme, which was detected in an *A. baumannii* isolate from the UK in 1985 [3]. Subsequently, several CHDLs have been reported, mostly in *Acinetobacter* spp. However, the most prevalent CHDL in Enterobacteriales, OXA-48, was reported in 2001 in Turkey from a clinical *K. pneumoniae* isolate [3,13]. Class D carbapenemases are serine enzymes that are resistant to inhibition by the commercially available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam), although they are inhibited in vitro by NaCl [124]. Notably, despite their significant activity, all class D carbapenemases do not confer a high level of carbapenem resistance owing to their weak carbapenem-hydrolysing activity [19,124].

#### 3.3.1. Enterobacteriales

The most reported CHDL among Enterobacteriales isolated from different aqueous environments is the phantom menace, the OXA-48 enzyme (Table 3; Fig. 4). OXA-48-producers belonging to different Enterobacteriales genera and species including *Citrobacter* spp., *E. coli*, *K. pneumoniae*, *K. oxytoca*, *C. freundii*, *C. braakii*, *Citrobacter youngae*, *C. farmeri*, *E. cloacae*, *E. aerogenes*, *E. kobei*, *S. marcescens*, *P. rettgeri*, *R. ornithinolytica* and *C. malonaticus* have been cultivated from wastewater and WWTPs, hospital sewage, puddles, rivers, estuaries, spring water, irrigation water, fountain water, seawater, water dam and drinking water in different parts of the world [27,32,39,46,63,75,92,97,108,125–130]. Although OXA-48-type is the most prevalent, other variants were reported among different Enterobacteriales. OXA-48-like-producing *K. oxytoca* has been isolated from hospital sewage in Algeria [131]. More recently, the *bla*<sub>OXA-48-like</sub> gene was detected in *K. pneumoniae* and *E. coli* strains isolated from seawater in Ireland [132]. In addition, Antonelli et al. have reported the isolation of OXA-372-producing *C. freundii* from hospital sewage in Italy [133]. Furthermore, OXA-370-producing *Citrobacter* sp. was recovered from seawater in Brazil. OXA-181-producing *E. coli* and *K. pneumoniae* were cultivated from hospital sewage in Switzerland and from a WWTP in the UK, respectively [46,63]. In addition, OXA-181-producing *E. coli* has been isolated from drinking water in the USA [128]. Another OXA-48-like variant, namely OXA-204, has been detected in a *C. braakii* isolate recovered from a WWTP in Tunisia [134]. Furthermore, OXA-244-producing *E. coli* isolates were obtained from river water in Algeria and from estuaries in Lebanon [126,127]. In China, Xin et al. reported the isolation of OXA-58-producing *Raoultella* from seawater [135]. More recently, OXA-655- and OXA-656-producing *E. coli*
<table><caption>**Table 3**
Epidemiology of class D carbapenemase-producers detected in aquatic environments</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="12">OXA-23</td><td rowspan="2">Algeria</td><td>Hospital sewage</td><td>Acinetobacter baumannii (2)</td><td>–</td><td>–</td><td>[131]</td></tr><tr><td>Hospital sewage</td><td>Acinetobacter baumannii (3)</td><td>–</td><td>–</td><td>[138]</td></tr><tr><td rowspan="2">Brazil</td><td>Seawater</td><td>Acinetobacter spp. (11)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>Hospital sewage</td><td>Acinetobacter baumannii (2)</td><td>–</td><td>–</td><td>[139]</td></tr><tr><td rowspan="4">Croatia</td><td>Liquid manure</td><td>Acinetobacter baumannii (2)</td><td>ST195</td><td>–</td><td>[147]</td></tr><tr><td rowspan="3">WWTP</td><td>Acinetobacter baumannii (10)</td><td>–</td><td>–</td><td>[141]</td></tr><tr><td>Acinetobacter baumannii (1)</td><td>–</td><td>–</td><td>[143]</td></tr><tr><td>Acinetobacter baumannii (10)</td><td>ST2</td><td>–</td><td>[145]</td></tr><tr><td rowspan="4">France</td><td>River</td><td>Acinetobacter baumannii (1)</td><td>–</td><td>–</td><td>[137]</td></tr><tr><td>River</td><td>Acinetobacter baumannii (2)</td><td>–</td><td>–</td><td>[120]</td></tr><tr><td>River</td><td>Acinetobacter baumannii (2)</td><td>–</td><td>–</td><td>[119]</td></tr><tr><td>Seawater</td><td>Acinetobacter spp. (6)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td rowspan="4">OXA-24/40</td><td>Romania</td><td>WWTP</td><td>Acinetobacter baumannii (3)</td><td>–</td><td>–</td><td>[141]</td></tr><tr><td>Croatia</td><td>Surface water</td><td>Acinetobacter baumannii (1)</td><td>–</td><td>–</td><td>[146]</td></tr><tr><td>Nigeria</td><td>River</td><td>Acinetobacter baumannii (1)</td><td>–</td><td>–</td><td>[119]</td></tr><tr><td>Brazil</td><td>Fountain water</td><td>Raoultella ornithinolytica (1)</td><td>–</td><td>IncL</td><td>[129]</td></tr><tr><td rowspan="12">OXA-48</td><td rowspan="2">Serbia</td><td>River</td><td>Klebsiella pneumoniae (1)</td><td>ST1393</td><td></td><td></td></tr><tr><td>Irrigation water</td><td>Klebsiella pneumoniae (3)</td><td>ST133, ST2055, ST2192</td><td>–</td><td>[126]</td></tr><tr><td rowspan="4">Algeria</td><td>River</td><td>Raoultella ornithinolytica (3), Citrobacter braakii (1), Citrobacter freundii (1) Escherichia coli (9)</td><td>–</td><td></td><td></td></tr><tr><td></td><td>ST559, ST38, ST212, ST1972, ST2142</td><td></td><td></td><td></td></tr><tr><td></td><td>Klebsiella pneumoniae (1) Escherichia coli (2)</td><td>ST13, ST5, ST19</td><td>IncL</td><td>[129]</td></tr><tr><td>Seawater</td><td>Raoultella ornithinolytica (1)</td><td>–</td><td></td><td></td></tr><tr><td rowspan="2">Spring water</td><td>Cronobacter malonaticus (1)</td><td>–</td><td></td><td></td></tr><tr><td>Dam water</td><td>Cronobacter malonaticus (1)</td><td>–</td><td></td><td></td></tr><tr><td rowspan="4">Austria</td><td>WWTP</td><td>Klebsiella pneumoniae (1)</td><td>ST1878</td><td></td><td></td></tr><tr><td>WWTP</td><td>Klebsiella pneumoniae (2)</td><td>ST1393, ST13</td><td></td><td></td></tr><tr><td></td><td>Enterobacter cloacae (1)</td><td>ST527</td><td></td><td></td></tr><tr><td></td><td>Klebsiella pneumoniae (1)</td><td>ST35</td><td></td><td></td></tr><tr><td rowspan="2">Belgium</td><td>Hospital WW (sinks)</td><td>Escherichia coli (1)</td><td>ST38</td><td>–</td><td>[39]</td></tr><tr><td>DDW</td><td>Klebsiella pneumoniae (1)</td><td>ST15</td><td>–</td><td></td></tr><tr><td>Egypt</td><td>Clinical and urban WW</td><td>Citrobacter freundii (5)</td><td>–</td><td>–</td><td>[130]</td></tr><tr><td>Germany</td><td>DDW</td><td>Klebsiella pneumoniae (2)</td><td>–</td><td>–</td><td>[27]</td></tr><tr><td rowspan="2">India</td><td>Clinical and urban WW</td><td>Klebsiella pneumoniae (3)</td><td>ST253, ST11</td><td>–</td><td>[76]</td></tr><tr><td>WWTP</td><td>Escherichia coli (3)</td><td>–</td><td>–</td><td>[108]</td></tr><tr><td rowspan="2">Morocco</td><td>Hospital sewage</td><td>Citrobacter braakii (2), Citrobacter farmeri (1)</td><td>–</td><td>IncFLC, IncP, IncHI1</td><td>[75]</td></tr><tr><td>Puddles</td><td>Enterobacter cloacae complex (6), Citrobacter freundii (3)</td><td>–</td><td>–</td><td>[32]</td></tr><tr><td rowspan="4">Philippines</td><td>River</td><td>Klebsiella pneumoniae (2)</td><td>ST3145, ST323</td><td>–</td><td></td></tr><tr><td>River</td><td>Klebsiella oxytoca (1)</td><td>ST95</td><td>–</td><td></td></tr><tr><td>River</td><td>Escherichia coli (1)</td><td>ST354</td><td>IncL</td><td>[127]</td></tr><tr><td>River</td><td>Klebsiella pneumoniae (1)</td><td>ST16</td><td>IncL</td><td></td></tr><tr><td rowspan="2">Portugal</td><td>Hospital sewage</td><td>Serratia marcescens (2)</td><td>–</td><td>–</td><td>[125]</td></tr><tr><td>River</td><td>Escherichia coli (1)</td><td>–</td><td>–</td><td>[64]</td></tr><tr><td rowspan="2">Romania</td><td>River</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[140]</td></tr><tr><td>Hospital sewage</td><td>Klebsiella pneumoniae (12)</td><td>–</td><td>–</td><td>[97]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 3 (continued)**</caption><thead><tr><th>Carbapenemase</th><th>Country</th><th>Source</th><th>Host(s) (n)</th><th>ST</th><th>Plasmid group</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">OXA-48-like</td><td rowspan="3">Switzerland</td><td>Hospital sewage</td><td>Escherichia coli (5)</td><td>ST215, ST38, ST393</td><td>–</td><td rowspan="10">[46]</td></tr><tr><td rowspan="2">WWTP</td><td>Klebsiella pneumoniae (3)</td><td>ST437, ST258</td><td>–</td></tr><tr><td>Enterobacter cloacae (1), Citrobacter freundii (6), Citrobacter youngae (1), Citrobacter spp. (2)</td><td>–</td><td>–</td></tr><tr><td rowspan="4">UK</td><td>Escherichia coli (9)</td><td>ST38, ST10, ST354, ST215</td><td>–</td></tr><tr><td rowspan="3">USA</td><td>Klebsiella pneumoniae (3)</td><td>ST485, ST395, ST29</td><td>–</td></tr><tr><td>Citrobacter freundii (3), Citrobacter sp. (1)</td><td>–</td><td>–</td></tr><tr><td>Raoultella ornithinolytica (2), Klebsiella oxytoca (1), Enterobacter kobei (1)</td><td>–</td><td>IncL/M</td><td>[63]</td></tr><tr><td rowspan="2">Ireland</td><td>Escherichia coli (1), Providencia rettgeri (1) Shewanella (2), Pandoraea sputorum (1), Pseudomonas (1)</td><td>–</td><td>–</td><td rowspan="2">[128]</td></tr><tr><td>Klebsiella oxytoca (2)</td><td>–</td><td>–</td></tr><tr><td rowspan="4">OXA-58</td><td>Algeria</td><td>Hospital sewage</td><td>Escherichia coli (1)</td><td>ST131</td><td>–</td><td rowspan="4">[132]</td></tr><tr><td rowspan="2">Ireland</td><td>Seawater</td><td>Klebsiella pneumoniae (1)</td><td>ST101</td><td>–</td></tr><tr><td>Enterobacter kobei (1)</td><td>Aeromonas caviae (1)</td><td>–</td><td>–</td></tr><tr><td>Singapore</td><td>Hospital sewage</td><td>Acinetobacter spp. (13)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td rowspan="5">Brazil</td><td>Seawater</td><td>Acinetobacter spp. (7)</td><td>–</td><td>–</td><td>[96]</td></tr><tr><td rowspan="5">China</td><td>Hospital sewage</td><td>Pseudomonas (3), Stenotrophomonas (1), Rheinheimera (2), Shewanella (1), Pseudoalteromonas (1), Algoriphagus (1), Bowmanella (1), Thalassospira (1), Raoultella (1), Vibrio (1)</td><td>–</td><td>–</td><td>[135]</td></tr><tr><td>Seawater</td><td>Acinetobacter lwooffii (1), Enterobacter cloacae (1)</td><td>–</td><td>–</td><td>[108]</td></tr><tr><td rowspan="2">WWTP</td><td>Acinetobacter baumannii (2)</td><td>ST1</td><td>–</td><td>[145]</td></tr><tr><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>Algeria</td><td>River</td><td>Escherichia coli (1)</td><td>–</td><td>–</td><td>[144]</td></tr><tr><td>Philippines</td><td>Hospital sewage</td><td>Escherichia coli (3)</td><td>ST940, ST410</td><td>–</td><td>[46]</td></tr><tr><td rowspan="2">Switzerland</td><td rowspan="2">WWTP</td><td>Klebsiella pneumoniae (1)</td><td>–</td><td>–</td><td>[63]</td></tr><tr><td>Escherichia coli (1)</td><td>–</td><td>–</td><td>[128]</td></tr><tr><td>UK</td><td>WWTP</td><td>Acinetobacter baumannii complex (1)</td><td>–</td><td>–</td><td>[63]</td></tr><tr><td>USA</td><td>DW</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[144]</td></tr><tr><td rowspan="2">OXA-199</td><td>Algeria</td><td>River</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[144]</td></tr><tr><td>Tunisia</td><td>WWTP</td><td>Citrobacter braakii (1)</td><td>–</td><td>–</td><td>[134]</td></tr><tr><td>OXA-204</td><td>Brazil</td><td>River</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[140]</td></tr><tr><td>OXA-181</td><td>Algeria</td><td>River</td><td>Escherichia coli (3)</td><td>ST3541</td><td>–</td><td>[126]</td></tr><tr><td>Lebanon</td><td>Estuaries</td><td>Escherichia coli (2)</td><td>–</td><td>ST38</td><td>IncHI2</td><td>[127]</td></tr><tr><td>OXA-252</td><td>USA</td><td>DW</td><td>Shewanella (1), Pseudomonas (1), Pseudomonas putida (1)</td><td>–</td><td>–</td><td>[128]</td></tr><tr><td>OXA-370</td><td>Brazil</td><td>Seawater</td><td>Citrobacter sp. (1)</td><td>–</td><td>–</td><td>[48]</td></tr><tr><td>OXA-372</td><td>Italy</td><td>Hospital sewage</td><td>Citrobacter freundii (1)</td><td>–</td><td>IncA/C, IncN</td><td>[133]</td></tr><tr><td>OXA-416</td><td>Algeria</td><td>Hospital sewage</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[141]</td></tr><tr><td>OXA-538</td><td>Algeria</td><td>River</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[144]</td></tr><tr><td>OXA-655</td><td>Brazil</td><td>Hospital sewage</td><td>Escherichia coli (1)</td><td>ST401</td><td>IncQ1</td><td>[136]</td></tr><tr><td>OXA-656</td><td>China</td><td>Animal sewage</td><td>Enterobacter cloacae (1)</td><td>ST24</td><td>IncQ1</td><td>[136]</td></tr><tr><td>OXA-894</td><td>China</td><td>Animal sewage</td><td>Shewanella xiamenensis (1)</td><td>–</td><td>–</td><td>[148]</td></tr></tbody></table>

n, number of strains; ST, sequence type; WWTP, wastewater treatment plant; WW, wastewater; DW, drinking water.
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Phylogenetic tree of class D carbapenemase variants detected in different aquatic environments. The evolutionary history was inferred using the neighbour-joining method. Evolutionary distances were computed using the Kimura 2-parameter method. The number above the nodes is the level of bootstrap from 1000 replicates.</figcaption></figure>

and *E. cloacae* were isolated from hospital waste in Brazil [136]. Although mainly reported in *Acinetobacter* species, the OXA-58 carbapenemase was detected in an *E. cloacae* isolate recovered from a WWTP in Germany [108].

### 3.3.2. Other Gram-negative bacilli

The first OXA-23-producing *A. baumannii* environmental isolate was obtained from the Seine river in downtown Paris. The *bla*OXA-23 gene was chromosomally encoded and pulse-field gel electrophoresis (PFGE) revealed that the isolate was clonally related to a previously identified human isolate obtained in New Caledonia in June 2004 [137]. Thereafter, OXA-23-producing *A. baumannii* isolates were recovered from hospital sewage in Brazil [138] and Croatia [139]. Subsequently, different studies have reported the detection of CHDLs in different water habitats. OXA-48- and OXA-204-producing *Shewanella xiamenensis* isolates were obtained from a Portuguese river in 2013 [140]. In 2015, Koh et al. reported the isolation of an OXA-48-type carbapenemase-producing *A. caviae* from hospital sewage in Singapore [74]. In addition, OXA-23- and OXA-40/24-producing *A. baumannii* isolates were obtained from a WWTP in Croatia in 2016 [141]. In the same year, Yousfi et al. reported the isolation of OXA-416-producing *S. xiamenensis* from hospital sewage in Algeria [142]. In 2017, OXA-23- and OXA-24/40-producing *A. baumannii* isolates were obtained from river water in Romania and Serbia [120]. In the same year, Goic-Barisic et al. described the detection of OXA-23-producing *A. baumannii* in a WWTP in Croatia [143]. In another study published in 2017, OXA-23-, OXA-24/40-, OXA-51-, OXA-58- and OXA-143-producing *Acinetobacter* spp. were isolated from seawater in Brazil [48].

In 2018, only three studies reported the detection of CHDL-producing glucose-non-fermenting GNB in a water environment. The first report described the isolation of *S. xiamenensis* isolates producing OXA-181, OXA-199 and a new variant, OXA-538, from

river water in Algeria [144]. The second report documented the isolation of OXA-23-producing *A. baumannii* from hospital sewage also in Algeria [131]. The third study was from Croatia where the authors have reported the isolation of OXA-23- and OXA-72-producing *A. baumannii* isolates from a WWTP [145].

Recently, OXA-58-producing isolates belonging to different genera, namely *Pseudomonas* spp., *Rheinheimera* spp., *Stenotrophomonas* spp., *Shewanella* spp., *Vibrio*, *Pseudoalteromonas*, *Algoriphagus*, *Bowmanella* and *Thalassospira*, were obtained from seawater in China [135]. In addition, Tacão et al. reported the isolation of OXA-48-producing *Shewanella* spp., *Pseudomonas* spp. and *Pandoraea sputorum*, OXA-181-producing *A. baumannii* complex and OXA-252-producing *Shewanella* sp. from drinking water in the USA [140]. Recently, OXA-40-producing *A. baumannii*, OXA-58-producing *Acinetobacter* spp. and *Acinetobacter lwoffii* were detected in surface water, hospital sewage and a WWTP, respectively [96,108,146].

Animal waste has also been reported as a reservoir of carbapenemase-producing isolates. Hrenovic et al. reported the isolation of OXA-23-producing *A. baumannii* ST195 from liquid manufacture in Croatia [147]. Furthermore, the new OXA-48-like variant OXA-894 was first described in a *S. xiamenensis* isolated from pig wastewater [148].

### 3.4. Carbapenemase-encoding genes detected by culture-independent methods

In addition to culture-based techniques, culture-independent methods such as quantitative real-time PCR and metagenomics are increasingly used in studying antibiotic resistance in the environment [149]. With regard to carbapenemase determinants, the *bla* genes encoding carbapenemases of Ambler classes A, B and D were detected in different water environments worldwide. Regarding
class A carbapenemases, *bla*<sub>KPC</sub> was detected in hospital sewage in Spain [150], Tunisia [151], Germany [76], Belgium [152] and India [153]. The KPC-encoding gene was also detected in rivers in Spain [150], India [154], Brazil [26,155], China [156] and Belgium [152], streams and lagoon water in Brazil [26,42], lake water in Poland [157] and Brazil [158], a fishpond and pig wastewater in China [159,160], municipal wastewater in India [154], Germany [161] and Belgium [152], and drinking water from a first nation community in Canada [162] and Brazil [155]. The *bla*<sub>GES-5</sub> and *bla*<sub>GES-16</sub> carbapenemase-encoding genes were also detected by culture-independent methods in WWTP and lagoon water, respectively [42,161]. In addition, *bla*<sub>IMI</sub> was detected in lake water in Poland [157] and in river sediments in China [163]. Finally, *bla*<sub>SFC-1</sub> was detected in lake water in Sweden [164].

Among the class B carbapenemase determinants, *bla*<sub>GIM</sub>, *bla*<sub>NDM</sub>, *bla*<sub>IMP</sub>, *bla*<sub>SPM</sub> and *bla*<sub>VIM</sub> genes were detected in different water habitats, including hospital sewage, wastewater and WWTPs, animal waste, rivers, lakes, rice field, drinking water and drinking water treatment plants. Detection of these MBL-encoding genes in the abovementioned environments was reported from many countries, namely Spain [150], Tunisia [151], China [43,156,165-168], India [153,154,169,170], Italy [171], Brazil [26,42,155,158], Canada [162,172], Switzerland [173], Belgium [152], Singapore [174], the USA [158], Poland [157,175], Germany [176] and Sweden [164].

Regarding class D carbapenemases, *bla*<sub>OXA-48</sub> determinants were detected by culture-independent methods in hospital sewage, wastewater and WWTPs, rivers, creeks, lakes, rice field and drinking water from Spain [150], Germany [176], Tunisia [151], India [153,154], Brazil [26,158], Belgium [152], Canada [162,172], Portugal [177], Poland [175] and Sweden [164,178]. In addition, the OXA-23-encoding gene has been detected in hospital sewage in Sweden [164]. Recently, *bla*<sub>OXA-58</sub> was detected in drinking water in Germany [179] and hospital sewage in India [153].

## 4. Multilocus sequence typing (MLST) analysis

MLST is a widely used method for typing bacterial strains. It was described in 1998 and consists of the examination of nucleotide sequences of seven housekeeping gene fragments of approximately 500 nucleotides. Since then, this method has been used for different purposes including epidemiological surveillance and population analysis [180,181].

Among carbapenemase-producing bacteria isolated from aquatic environments, *K. pneumoniae*, *E. coli* and *E. cloacae* are the most characterised species by MLST. PHYLOViZ Online [182] was used for conducting phylogenetic analysis of sequence types (STs) of *K. pneumoniae*, *E. coli* and *E. cloacae* detected in aquatic environments. Our analysis revealed a remarkable diversity of STs detected, especially regarding *K. pneumoniae* and *E. coli*. Phylogenetic analysis of *K. pneumoniae*, *E. coli* and *E. cloacae* STs with the harboured carbapenemase class and their geographical areas are presented in Fig. 5. Regarding *K. pneumoniae*, all of the major epidemic high-risk international clones, namely ST11, ST15, ST101, ST147 and ST258 [183], have been detected in different countries. Of note, carbapenemases of Ambler classes A and D have been detected in ST258, known to be the most globally disseminated. Regarding *E. coli*, several international high-risk clones such as ST38, ST131, ST410 and ST648 [184] have been detected in water environments. ST38 has been largely associated with Ambler class D carbapenemases (OXA-48). This was also the case in water isolates, where all *E. coli* ST38 isolates harboured the *bla*<sub>OXA-48</sub> gene as shown in Fig. 5. In contrast, STs of *E. cloacae* are mostly associated with Ambler class A carbapenemases and, unlike *K. pneumoniae* and *E. coli*, none of the most prevalent and widespread *E. cloacae* clones were detected in aquatic environments.

In addition, we investigated the isolation of carbapenemase-producing *K. pneumoniae*, *E. coli* and *E. cloacae* clones detected in water environments from human infections in the respective countries and the results are also shown in Fig. 5. Several carbapenemase-producing clones of *K. pneumoniae* and *E. coli* have been reported to cause human infections in countries where they were detected in aquatic environments [83,185-199]. Indeed, detection of the same clones with the same resistance mechanism in the same geographical area both in clinical and water isolates might be of great importance. This foretells the danger that the presence of these organisms in water can cause and suggests the potential participation of aquatic environments in the dissemination of these bacteria.

## 5. Conclusion

Carbapenems are among our last-resort antibiotics against drug-resistant pathogens, making carbapenem resistance a great health concern, especially that due to carbapenemase production. Knowledge of environmental reservoirs of resistant organisms and resistance genes is crucial in our quest to control their dissemination. The data presented here confirm the wide dissemination of carbapenemase-producers and carbapenemase-encoding genes in the natural environment and other water habitats, presenting a serious problem for human and animal health. In this review, we aimed to give an overview of the state of the art regarding the spread of carbapenemase-producing bacteria in different aquatic environments, which may help in implementing prevention and control strategies. Indeed, the interconnectedness between the environment and the health of humans, animals and plants makes the surveillance and control of the antibiotic resistance phenomenon a very difficult task. Hence the urgent need for an interdisciplinary collaboration to establish effective control and prevention strategies against the spread of carbapenemase-producing bacteria. In this context, the US National Action Plan for Combating Antibiotic-Resistant Bacteria (CARB) was created and will be followed over 5 years (2020-2025) in order to change the course of antibiotic resistance [https://aspe.hhs.gov/system/files/pdf/264126/CARB-National-Action-Plan-2020-2025.pdf]. The main goals of CARB are (i) slowing the emergence of resistant bacteria and preventing the spread of resistant infections, (ii) strengthening 'One Health' surveillance efforts to combat bacterial resistance, (iii) development and use of rapid and innovative diagnostic tools, (iv) development of new antibiotics, other therapies and vaccines and (v) improving international collaboration for prevention, surveillance, control, research and development of antibiotics. This plan integrates in parallel a 'One Health' approach with special emphasis on understanding antibiotic resistance in the environment. The application of such plans in other countries could help in the control of spread of drug-resistant bacteria. On the other hand, the application of obligatory reporting of antibiotic resistance in veterinary and human clinical settings, and possibly in water treatment facilities, will enable countries that do not have such surveillance plans assess contemporary prevention measures and target the areas of greatest concern [200]. In addition, it seems clear that wastewater, whatever its origin, is the main reservoir of resistant bacteria among all aquatic environments. Consequently, it should be a primary target for control and prevention efforts. Thus, developing effective wastewater treatment methods for removing or at least decreasing antibiotic-resistant bacteria and antibiotic resistance genes in the final effluent is strongly recommended.

**Acknowledgments:** This work was supported by the French Government under the 'Investments for the Future' program man-
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Multilocus sequence typing (MLST) data set generated using PHYLOViZ Online, indicating the sequence types (STs) of carbapenemase-producing *Klebsiella pneumoniae*, *Escherichia coli* and *Enterobacter cloacae* detected in aquatic environments with the respective carbapenemase class and geographical area.</figcaption></figure>

aged by the National Agency for Research (ANR) [Méditerranée-Infection 10-IAHU-03].

Competing interests: None declared.

Ethical approval: Not required.

## References

[1] Tacconelli E, Carrara E, Savoldi A, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 2018;18:318-27.

[2] Papp-Wallace KM, Endimiani A, Taracila MA, et al. Carbapenems: past, present, and future. Antimicrob Agents Chemother 2011;55:4943-60.

[3] Potter RF, D'Souza AW, Dantas G. The rapid spread of carbapenem-resistant Enterobacteriaceae. Drug Resist Updat 2016;29:30-46.

[4] Maltezou HC. Metallo-β-lactamases in Gram-negative bacteria: introducing the era of pan-resistance? Int J Antimicrob Agents 2009;33 405.e1-7.

[5] Diene SM, Rolain JM. Carbapenemase genes and genetic platforms in Gram-negative bacilli: Enterobacteriaceae, Pseudomonas and Acinetobacter species. Clin Microbiol Infect 2014;20:831-8.

[6] Nordmann P, Dortel L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm!. Trends Mol Med 2012;18:263-72.

[7] Bakthavatchalam YD, Anandan S, Veeraraghavan B. Laboratory detection and clinical implication of oxacillinase-48 like carbapenemase: the hidden threat. J Glob Infect Dis 2016;8:41-50.

[8] Jean SS, Lee WS, Lam C, et al. Carbapenemase-producing Gram-negative bacteria: current epidemics, antimicrobial susceptibility and treatment options. Future Microbiol 2015;10:407-25.

[9] Berglund B. Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics. Infect Ecol Epidemiol 2015;5:28564.

[10] Vaz-Moreira I, Nunes OC, Manaia CM. Bacterial diversity and antibiotic resistance in water habitats: searching the links with the human microbiome. FEMS Microbiol Rev 2014;38:761-78.

[11] Zhang XX, Zhang T, Fang HH. Antibiotic resistance genes in water environment. Appl Microbiol Biotechnol 2009;82:397-414.

[12] Manaia CM, Macedo G, Fatta-Kassinos D, et al. Antibiotic resistance in urban aquatic environments: can it be controlled? Appl Microbiol Biotechnol 2016;100:1543-57.

[13] Tamma PD, Simner PJ. Phenotypic detection of carbapenemase-producing organisms from clinical isolates. J Clin Microbiol 2018;56:e011470-18.

[14] Bush K, Jacoby GA. Updated functional classification of β-lactamases. Antimicrob Agents Chemother 2010;54:969-76.

[15] Yoo JH. The Infinity War: how to cope with carbapenem-resistant Enterobacteriaceae. J Korean Med Sci 2018;33:e255.

[16] Oueslati S, Girlich D, Dortel L, et al. Evaluation of the Amplidiag CarbaR+VRE Kit for accurate detection of carbapenemase-producing bacteria. J Clin Microbiol 2018;56:e01092-17.

[17] Walther-Rasmussen J, Hoiby N. Class A carbapenemases. J Antimicrob Chemother 2007;60:470-82.

[18] Iovleva A, Doi Y. Carbapenem-resistant Enterobacteriaceae. Clin Lab Med 2017;37:303-15.

[19] Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect 2014;20:821-30.

[20] Henriques I, Moura A, Alves A, et al. Molecular characterization of a carbapenem-hydrolyzing class A β-lactamase, SFC-1, from Serratia fonticola UTAD54. Antimicrob Agents Chemother 2004;48:2321-4.

[21] Aubron C, Poirel L, Ash RJ, et al. Carbapenemase-producing Enterobacteriaceae, U.S. rivers. Emerg Infect Dis 2005;11:260-4.

[22] Harmon DE, Miranda OA, McCarley A, et al. Prevalence and characterization of carbapenem-resistant bacteria in water bodies in the Los Angeles-Southern California area. Microbiologyopen 2018;8:e00692.

[23] Laurens C, Jean-Pierre H, Licznar-Fajardo P, et al. Transmission of IMI-2 carbapenemase-producing Enterobacteriaceae from river water to human. J Glob Antimicrob Resist 2018;15:88-92.

[24] Piedra-Carrasco N, Fabrega A, Calero-Caceres W, et al. Carbapenemase-producing Enterobacteriaceae recovered from a Spanish river ecosystem. PLoS One 2017;12:e0175246.

[25] Montezzi LF, Campana EH, Correa LL, et al. Occurrence of carbapenemase-producing bacteria in coastal recreational waters. Int J Antimicrob Agents 2015;45:174-7.

[26] Sanchez DG, de Melo FM, Savazzi EA, et al. Detection of different β-lactamases encoding genes, including blaNDM, and plasmid-mediated quinolone resistance genes in different water sources from Brazil. Environ Monit Assess 2018;190:407.

[27] Hamza E, Dorgham SM, Hamza DA. Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt. J Glob Antimicrob Resist 2016;7:8-10.

[28] Haller L, Chen H, Ng C, et al. Occurrence and characteristics of extended-spectrum β-lactamase- and carbapenemase-producing bacteria from hospital effluents in Singapore. Sci Total Environ 2018;615:1119-25.

[29] Weingarten RA, Johnson RC, Conlan S, et al. Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance. mBio 2018;9:e02011-17.

[30] Hoelle J, Johnson JR, Johnston B, et al. Survey of US wastewater for carbapenem-resistant Enterobacteriaceae. J Water Health 2019;17:219-26.

[31] Haberecht HB, Nealon NJ, Gilliland JR, et al. Antimicrobial-resistant *Escherichia*
coli from environmental waters in Northern Colorado. J Environ Public Health 2019;2019:3862949.

[32] Cahill N, O'Connor L, Mahon B, et al. Hospital effluent: a reservoir for carbapenemase-producing Enterobacterales? Sci Total Environ 2019;672:618–24.

[33] Sakkas H, Bozidis P. Antimicrobial resistance in bacterial pathogens and detection of carbapenemases in Klebsiella pneumoniae isolates from hospital wastewater. Antibiotics (Basel) 2019;8:85.

[34] Swedan S, Abu Alrub H. Antimicrobial resistance, virulence factors, and pathotypes of Escherichia coli isolated from drinking water sources in Jordan. Pathogens 2019;8:86.

[35] Jung J, Choi HS, Lee JY, et al. Outbreak of carbapenemase-producing Enterobacteriaceae associated with a contaminated water dispenser and sink drains in the cardiology units of a Korean hospital. J Hosp Infect 2020;104:476–83.

[36] Poirel L, Barbosa-Vasconcelos A, Simoes RR, et al. Environmental KPC-producing Escherichia coli isolates in Portugal. Antimicrob Agents Chemother 2012;56:1662–3.

[37] Chagas TP, Seki LM, da Silva DM, et al. Occurrence of KPC-2-producing Klebsiella pneumoniae strains in hospital wastewater. J Hosp Infect 2011;77:281.

[38] Zhang X, Lu X, Zong Z. Enterobacteriaceae producing the KPC-2 carbapenemase from hospital sewage. Diagn Microbiol Infect Dis 2012;73:204–6.

[39] Galler H, Feierl G, Petternel C, et al. KPC-2 and OXA-48 carbapenemase-harbouring Enterobacteriaceae detected in an Austrian wastewater treatment plant. Clin Microbiol Infect 2014;20:0132–4.

[40] Picoa RC, Cardoso JP, Campana EH, et al. The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage. Diagn Microbiol Infect Dis 2013;76:80–5.

[41] Oliveira S, Moura RA, Silva KC, et al. Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers. J Antimicrob Chemother 2014;69:849–52.

[42] de Araujo CF, Silva DM, Carneiro MT, et al. Detection of carbapenemase genes in aquatic environments in Rio de Janeiro, Brazil. Antimicrob Agents Chemother 2016;60:4380–3.

[43] Yang F, Mao D, Zhou H, et al. Prevalence and fate of carbapenemase genes in a wastewater treatment plant in Northern China. PLoS One 2016;11:e0156383.

[44] Kittinger C, Lipp M, Folli B, et al. Enterobacteriaceae isolated from the River Danube: antibiotic resistances, with a focus on the presence of ESBL and carbapenemases. PLoS One 2016;11:e0165820.

[45] Caltagirone M, Nucleo E, Spalla M, et al. Occurrence of extended spectrum β-lactamases, KPC-type, and MCR-1.2-producing Enterobacteriaceae from wells, river water, and wastewater treatment plants in Oltrepò Pavese Area. Northern Italy. Front Microbiol 2017;8:2232.

[46] Zurfluh K, Bagutti C, Brodmann P, et al. Wastewater is a reservoir for clinically relevant carbapenemase- and 16S rRNA methylase-producing Enterobacteriaceae. Int J Antimicrob Agents 2017;50:436–40.

[47] de Oliveira DV, Nunes LS, Barth AL, et al. Genetic background of β-lactamases in Enterobacteriaceae isolates from environmental samples. Microb Ecol 2017;74:599–607.

[48] Paschoal RP, Campana EH, Corra LL, et al. Concentration and variety of carbapenemase producers in recreational coastal waters showing distinct levels of pollution. Antimicrob Agents Chemother 2017;61:e01963-17.

[49] Xu H, Wang X, Yu X, et al. First detection and genomics analysis of KPC-2-producing Citrobacter isolates from river sediments. Environ Pollut 2018;235:931–7.

[50] Yang F, Huang L, Li L, et al. Discharge of KPC-2 genes from the WWTPs contributed to their enriched abundance in the receiving river. Sci Total Environ 2017;581–582:136–43.

[51] Sun P, Bi Z, Nilsson M, et al. Occurrence of blaKPC-2, blaCTX-M, and mcr-1 in Enterobacteriaceae from well water in rural China. Antimicrob Agents Chemother 2017;61:e02569-16.

[52] Jin L, Wang R, Wang X, et al. Emergence of mcr-1 and carbapenemase genes in hospital sewage water in Beijing. China. J Antimicrob Chemother 2018;73:84–7.

[53] Gomi R, Matsuda T, Yamamoto M, et al. Characteristics of carbapenemase-producing Enterobacteriaceae in wastewater revealed by genomic analysis. Antimicrob Agents Chemother 2018;62:e02501-17.

[54] Sekizuka T, Yatsu K, Inamine Y, et al. Complete genome sequence of a blaKPC-2-positive Klebsiella pneumoniae strain isolated from the effluent of an urban sewage treatment plant in. Japan. mSphere 2018;3:e00314-18.

[55] Yao Y, Lazaro-Perona F, Falghauer L, et al. Insights into a novel blaKPC-2-encoding IncP-6 plasmid reveal carbapenem-resistance circulation in several Enterobacteriaceae species from wastewater and a hospital source in Spain. Front Microbiol 2017;8:1143.

[56] Jelic M, Hrenovic J, Dekic S, et al. First evidence of KPC-producing ST258 Klebsiella pneumoniae in river water. J Hosp Infect 2019;103:147–50.

[57] Mathys DA, Mollenkopf DF, Feicht SM, et al. Carbapenemase-producing Enterobacteriaceae and Aeromonas spp. present in wastewater treatment plant effluent and nearby surface waters in the US. PLoS One 2019;14:e0218650.

[58] Perilli M, Bottoni C, Pontieri E, et al. Emergence of blaKPC-3-Tn4401a in Klebsiella pneumoniae ST512 in the municipal wastewater treatment plant and in the university hospital of a town in central Italy. J Glob Antimicrob Resist 2013;1:217–20.

[59] Teixeira P, Tacao M, Pureza L, et al. Occurrence of carbapenemase-producing

Enterobacteriaceae in a Portuguese river: blaNDM, blaKPC and blaGES among the detected genes. Environ Pollut 2020;260:113913.

[60] Manageiro V, Ferreira E, Canica M, et al. GES-5 among the β-lactamases detected in ubiquitous bacteria isolated from aquatic environment samples. FEMS Microbiol Lett 2014;351:64–9.

[61] White L, Hopkins KL, Meunier D, et al. Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates. J Hosp Infect 2016;93:145–51.

[62] Conte D, Palmeiro JK, da Silva Nogueira K, et al. Characterization of CTX-M enzymes, quinolone resistance determinants, and antimicrobial residues from hospital sewage, wastewater treatment plant, and river water. Ecotoxic Environ Saf 2017;136:62–9.

[63] Ludden C, Reuter S, Judge K, et al. Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MiniION sequencing. Microb Genom 2017;3:e000114.

[64] Suzuki Y, Nazareno PJ, Nakano R, et al. Environmental presence and genetic characteristics of carbapenemase-producing Enterobacteriaceae from hospital sewage and river water in the Philippines. Appl Environ Microbiol 2020;86:e01906-19.

[65] Mathers AJ, Vegesana K, German Mesner I, et al. Intensive care unit wastewater interventions to prevent transmission of multispecies Klebsiella pneumoniae carbapenemase-producing organisms. Clin Infect Dis 2018;67:171–8.

[66] Sekizuka T, Inamine Y, Segawa T, et al. Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan. Environ Microbiol Rep 2019;11:589–97.

[67] Girlich D, Poirel L, Nordmann P. Novel Ambler class A carbapenem-hydrolyzing β-lactamase from a Pseudomonas fluorescens isolate from the Seine River, Paris, France. Antimicrob Agents Chemother 2010;54:328–32.

[68] Neto WRN, Araujo JMM, da Silva DF, et al. Detection of Gram-negative bacteria carrying the blaKPC-2 gene from mangrove sediments. J Pharm Pharmacol 2019;7:485–92.

[69] Hammerl J, Jackel C, Bortolaia V, et al. Carbapenemase VCC-1-producing Vibrio cholerae in coastal waters of Germany. Emerg Infect Dis 2017;23:1735–7.

[70] Sabath LD, Abraham EP. Zinc as a cofactor for cephalosporinase from Bacillus cereus 569. Biochem J 1966;98 11C-3C.

[71] Ju LC, Cheng Z, Fast W, et al. The continuing challenge of metallo-β-lactamase inhibition: mechanism matters. Trends Pharmacol Sci 2018;39:635–47.

[72] Zahedi Bialvaei A, Samadi Kafil H, Ebrahizadeh Leylabadlo H, et al. Dissemination of carbapenemases producing Gram negative bacteria in the Middle East. Iran J Microbiol 2015;7:226–46.

[73] Potron A, Poirel L, Nordmann P. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: mechanisms and epidemiology. Int J Antimicrob Agents 2015;45:568–85.

[74] Koh TH, Ko K, Jureen R, et al. High counts of carbapenemase-producing Enterobacteriaceae in hospital sewage. Infect Control Hosp Epidemiol 2015;36:619–21.

[75] Parvez S, Khan AU. Hospital sewage water: a reservoir for variants of New Delhi metallo-β-lactamase (NDM)- and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Int J Antimicrob Agents 2018;51:82–8.

[76] Muller H, Sib E, Gajdiss M, et al. Dissemination of multi-resistant Gram-negative bacteria into German wastewater and surface waters. FEMS Microbiol Ecol 2018;94.

[77] Mahon BM, Brehony C, McGrath E, et al. Indistinguishable NDM-producing Escherichia coli isolated from recreational waters, sewage, and a clinical specimen in Ireland, 2016 to 2017. Euro Surveill 2017;22:30513.

[78] Walsh TR, Weeks J, Livermore DM, et al. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011;11:355–62.

[79] Shah TA, Zahra R. Screening of environment water for the presence of blaNDM-1 gene containing microorganisms. J Coll Physicians Surg Pak 2014;24:695–7.

[80] Chandran SP, Diwan V, Tamhankar AJ, et al. Detection of carbapenem resistance genes and cephalosporin, and quinolone resistance genes along with ogxAB gene in Escherichia coli in hospital wastewater: a matter of concern. J Appl Microbiol 2014;117:984–95.

[81] Toleman MA, Bugert JJ, Nizam SA. Extensively drug-resistant New Delhi metallo-β-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012. Emerg Infect Dis 2015;21:1027–30.

[82] Mantilla-Calderon D, Jumat MR, Wang T, et al. Isolation and characterization of NDM-positive Escherichia coli from municipal wastewater in Jeddah, Saudi Arabia. Antimicrob Agents Chemother 2016;60:5223–31.

[83] Zheng R, Zhang Q, Guo Y, et al. Outbreak of plasmid-mediated NDM-1-producing Klebsiella pneumoniae ST105 among neonatal patients in Yunnan, China. Ann Clin Microbiol Antimicrob 2016;15:10.

[84] Campana EH, Montezzi LF, Paschoal RP, et al. NDM-producing Klebsiella pneumoniae ST11 goes to the beach. Int J Antimicrob Agents 2017;49:119–21.

[85] Wu W, Espidoedo B, Feng Y, et al. Citrobacter freundii carrying blaKPC-2 and blaNDM-1: characterization by whole genome sequencing. Sci Rep 2016;6:30670.

[86] Yang F, Mao D, Zhou H, et al. Propagation of New Delhi metallo-β-lactamase genes (blaNDM-1) from a wastewater treatment plant to its receiving river. Environ Sci Technol Lett 2016;3:138–43.
[87] Akiba M, Sekizuka T, Yamashita A, et al. Distribution and relationships of antimicrobial resistance determinants among extended-spectrum-cephalosporin-resistant or carbapenem-resistant *Escherichia coli* isolates from rivers and sewage treatment plants in India. Antimicrob Agents Chemother 2016;60:2972–80.

[88] Zarfel G, Lipp M, Gurtl E, et al. Troubled water under the bridge: screening of River Mur water reveals dominance of CTX-M harboring *Escherichia coli* and for the first time an environmental VIM-1 producer in Austria. Sci Total Environ 2017;593–594:399–405.

[89] Islam MA, Islam M, Hasan R, et al. Environmental spread of New Delhi metallo-β-lactamase-1-producing multidrug-resistant bacteria in Dhaka, Bangladesh. Appl Environ Microbiol 2017;83:e00793–17.

[90] Daoud Z, Farah J, Sokhn ES, et al. Multidrug-resistant Enterobacteriaceae in Lebanese hospital wastewater: implication in the One Health concept. Microb Drug Resist 2018;24:166–74.

[91] Sugawara Y, Akeda Y, Hagiya H, et al. Spreading patterns of NDM-producing Enterobacteriaceae in clinical and environmental settings in Yangon, Myanmar. Antimicrob Agents Chemother 2019;63:e01924–18.

[92] Hmede Z, Sulaiman AAA, Jaafar H, et al. Emergence of plasmid-borne colistin resistance gene *mcr-1* in multidrug-resistant *Escherichia coli* isolated from irrigation water in Lebanon. Int J Antimicrob Agents 2019;54:102–4.

[93] Yang F, Gu Y, Zhou J, et al. Swine waste: a reservoir of high-risk *bla*NDM and *mcr-1*. Sci Total Environ 2019;683:308–16.

[94] Li Y, Luo L, Xiao Z, et al. Characterization of a carbapenem-resistant Kluvera crytocrescens isolate carrying *bla*NDM-1 from hospital sewage. Antibiotics (Basel) 2019;8:149.

[95] Bardhan T, Chakraborty M, Bhattacharjee B. Prevalence of colistin-resistant, carbapenem-hydrolyzing Proteobacteria in hospital water bodies and out-falls of West Bengal, India. Int J Environ Res Public Health 2020;17:1007.

[96] Zhang L, Ma X, Luo L, et al. The prevalence and characterization of extended-spectrum β-lactamase- and carbapenemase-producing bacteria from hospital sewage, treated effluents and receiving rivers. Int J Environ Res Public Health 2020;17:1182.

[97] Surleac M, Czobor Barbu I, Paraschiv S, et al. Whole genome sequencing snapshot of multi-drug resistant *Klebsiella pneumoniae* strains from hospitals and receiving wastewater treatment plants in Southern Romania. PLoS One 2020;15:e0228079.

[98] Zhai R, Fu B, Shi X, et al. Contaminated in-house environment contributes to the persistence and transmission of NDM-producing bacteria in a Chinese poultry farm. Environ Int 2020;139:105715.

[99] Isozumi R, Yoshimatsu K, Yamashiro T, et al. *bla*NDM-1-positive *Klebsiella pneumoniae* from environment. Vietnam. Emerg Infect Dis 2012;18:1383–5.

[100] Khan AU, Parvez S. Detection of *bla*NDM-4 in *Escherichia coli* from hospital sewage. J Med Microbiol 2014;63:1404–6.

[101] Long H, Feng Y, Ma K, et al. The co-transfer of plasmid-borne colistin-resistant genes *mcr-1* and *mcr-3.5*, the carbapenemase gene *bla*NDM-5 and the 16S methylase gene rmtB from *Escherichia coli*. Sci Rep 2019;9:696.

[102] Sekizuka T, Inamine Y, Segawa T, et al. Characterization of NDM-5- and CTX-M-55-coproducing *Escherichia coli* GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay. Infect Drug Resist 2019;12:2243–9.

[103] Di DY, Jang J, Unno T, et al. Emergence of Klebsiella variicola positive for NDM-9, a variant of New Delhi metallo-β-lactamase, in an urban river in South Korea. J Antimicrob Chemother 2017;72:1063–7.

[104] Kotsanas D, Wijesooriya WR, Korman TM, et al. Down the drain: carbapenem-resistant bacteria in intensive care unit patients and handwashing sinks. Med J Aust 2013;198:267–9.

[105] Vergara-Lopez S, Dominguez MC, Conejo MC, et al. Wastewater drainage system as an occult reservoir in a protracted clonal outbreak due to metallo-β-lactamase-producing Klebsiella oxytoca. Clin Microbiol Infect 2013;19:E490–8.

[106] Kieffer N, Poirel L, Bessa LJ, et al. VIM-1, VIM-34, and IMP-8 carbapenemase-producing *Escherichia coli* strains recovered from a Portuguese river. Antimicrob Agents Chemother 2016;60:2585–6.

[107] Khan FA, Hellmark B, Ehricht R, et al. Related carbapenemase-producing Klebsiella isolates detected in both a hospital and associated aquatic environment in Sweden. Eur J Clin Microbiol Infect Dis 2018;37:2241–51.

[108] Schages L, Wichern F, Kalscheuer R, et al. Winter is coming—impact of temperature on the variation of β-lactamase and mcr genes in a wastewater treatment plant. Sci Total Environ 2020;712:136499.

[109] Zurfluh K, Hachler H, Nuesch-Inderbinen M, et al. Characteristics of extended-spectrum β-lactamase- and carbapenemase-producing Enterobacteriaceae isolates from rivers and lakes in Switzerland. Appl Environ Microbiol 2013;79:3021–6.

[110] Saavedra MJ, Peixe L, Sousa JC, et al. Sfh-1, a subclass B2 metallo-β-lactamase from a *Serratia fonticola* environmental isolate. Antimicrob Agents Chemother 2003;47:2330–3.

[111] Suzuki Y, Ida M, Kubota H, et al. Multiple β-lactam resistance gene-carrying plasmid harbored by Klebsiella quasipneumoniae isolated from urban sewage in Japan. mSphere 2019;4:e00391–19.

[112] Sekiguchi J-i, Morita K, Kitao T, et al. KHM-1, a novel plasmid-mediated metallo-β-lactamase from a Citrobacter freundii clinical isolate. Antimicrob Agents Chemother 2008;52:4194.

[113] Zong Z, Zhang X. *bla*NDM-1-carrying *Acinetobacter* johnsonii detected in hospital sewage. J Antimicrob Chemother 2013;68:1007–10.

[114] Zhang C, Qiu S, Wang Y, et al. Higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals in Beijing. PLoS One 2014;8:e64857.

[115] Hu Y, Feng Y, Qin J, et al. *Acinetobacter* chinensis, a novel *Acinetobacter* species, carrying *bla*NDM-1, recovered from hospital sewage. J Microbiol 2019;57:350–5.

[116] Hu Y, Wu W, Feng Y, et al. Draft genome sequence of a *Pseudomonas* sp. strain carrying *bla*IMP-25 and *bla*VIM-2 carbapenemase genes from hospital sewage. Genome Announc 2016;4:e01027–16.

[117] Miranda CC, de Filippis I, Pinto LH, et al. Genotypic characteristics of multidrug-resistant *Pseudomonas* aeruginosa from hospital wastewater treatment plant in Rio de Janeiro, Brazil. J Appl Microbiol 2015;118:1276–86.

[118] Quinteira S, Ferreira H, Peixe L. First isolation of *bla*VIM-2 in an environmental isolate of *Pseudomonas* pseudoalcaligenes. Antimicrob Agents Chemother 2005;49:2140–1.

[119] Tacao M, Correia A, Henriques IS. Low prevalence of carbapenem-resistant bacteria in river water: resistance is mostly related to intrinsic mechanisms. Microb Drug Resist 2015;21:497–506.

[120] Kittinger C, Kirschner A, Lipp M, et al. Antibiotic resistance of *Acinetobacter* spp. isolates from the River Danube: susceptibility stays high. Int J Environ Res Public Health 2017;15:52.

[121] Adelowo OO, Vollmers J, Mausezahl I, et al. Detection of the carbapenemase gene *bla*VIM-5 in members of the *Pseudomonas* putida group isolated from polluted Nigerian wetlands. Sci Rep 2018;8:15116.

[122] Luo Y, Yang F, Mathieu J, et al. Proliferation of multidrug-resistant New Delhi metallo-β-lactamase genes in municipal wastewater treatment plants in Northern China. Environ Sci Techn Lett 2013;1:26–30.

[123] Oyelade AA, Adelowo OO, Fagade OE. *bla*NDM-1-producing Vibrio parahaemolyticus and V. vulnificus isolated from recreational beaches in Lagos, Nigeria. Environ Sci Pollut Res Int 2018;25:33538–47.

[124] Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012;67:1597–606.

[125] Potron A, Poirel L, Bussy F, et al. Occurrence of the carbapenem-hydrolyzing β-lactamase gene *bla*OXA-48 in the environment in Morocco. Antimicrob Agents Chemother 2011;55:4513–14.

[126] Taftouk R, Touati A, Leangapichart T, et al. Characterization of OXA-48-like-producing Enterobacteriaceae isolated from river water in Algeria. Water Res 2017;120:185–9.

[127] Diab M, Hamze M, Bonnet R, et al. Extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in water sources in Lebanon. Vet Microbiol 2018;217:97–103.

[128] Tanner WD, VanDerslice JA, Goel RK, et al. Multi-state study of Enterobacteriaceae harboring extended-spectrum β-lactamase and carbapenemase genes in U.S. drinking water. Sci Rep 2019;9:3938.

[129] Mairi A, Pantel A, Ousalem F, et al. OXA-48-producing Enterobacteriales in different ecological niches in Algeria: clonal expansion, plasmid characteristics and virulence traits. J Antimicrob Chemother 2019;74:1848–55.

[130] De Geyer D, Blommaert L, Verbraeken N, et al. The sink as a potential source of transmission of carbapenemase-producing Enterobacteriaceae in the intensive care unit. Antimicrob Resist Infect Control 2017;6:24.

[131] Yousfi K, Touati A, Lefebvre B, et al. Characterization of multidrug-resistant Gram-negative bacilli isolated from hospitals effluents: first report of a *bla*OXA-48-like in Klebsiella oxytoca, Algeria. Braz J Microbiol 2019;50:175–83.

[132] Mahon BM, Brehony C, Cahill N, et al. Detection of OXA-48-like-producing Enterobacteriales in Irish recreational water. Sci Total Environ 2019;690:1–6.

[133] Antonelli A, D'Andrea MM, Vaggelli G, et al. OXA-372, a novel carbapenem-hydrolysing class D β-lactamase from a Citrobacter freundii isolated from a hospital wastewater plant. J Antimicrob Chemother 2015;70:2749–56.

[134] Sghaier S, Abbassi MS, Pascual A, et al. Extended-spectrum β-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: first detection of O25b-B23-CTX-M-27-ST131 *Escherichia coli* and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater. J Glob Antimicrob Resist 2019;17:189–94.

[135] Xin R, Zhang K, Wu N, et al. The pollution level of the *bla*OXA-58 carbapenemase gene in coastal water and its host bacteria characteristics. Environ Pollut 2019;244:66–71.

[136] Kotsakis SD, Flach CF, Razavi M, et al. Characterization of the first OXA-10 natural variant with increased carbapenemase activity. Antimicrob Agents Chemother 2018;63:e01817–18.

[137] Girlich D, Poirel L, Nordmann P. First isolation of the *bla*OXA-23 carbapenemase gene from an environmental *Acinetobacter* baumannii isolate. Antimicrob Agents Chemother 2010;54:578–9.

[138] Ferreira AE, Marchetti DP, De Oliveira LM, et al. Presence of OXA-23-producing isolates of *Acinetobacter* baumannii in wastewater from hospitals in southern Brazil. Microb Drug Resist 2011;17:221–7.

[139] Bedenic B, Siroglavic M, Slade M, et al. Comparison of clinical and sewage isolates of *Acinetobacter* baumannii from two long-term care facilities in Zagreb: mechanisms and routes of spread. Arch Microbiol 2020;202:361–8.

[140] Tacão M, Correia A, Henriques I. Environmental Shewanella xiamenensis strains that carry *bla*OXA-48 or *bla*OXA-204 genes: additional proof for *bla*OXA-48-like gene origin. Antimicrob Agents Chemother 2013;57:6399–400.

[141] Goic-Barisic I, Hrenovic J, Kovacic A, et al. Emergence of oxacillinases in environmental carbapenem-resistant *Acinetobacter* baumannii associated with clinical isolates. Microb Drug Resist 2016;22:559–63.

[142] Yousfi K, Touati A, Lefebvre B, et al. A novel plasmid, pSx1, harboring a new Tn1696 derivative from extensively drug-resistant Shewanella xiamenensis encoding OXA-416. Microb Drug Resist 2017;23:429–36.
[143] Goic-Baricis I, Seruga Music M, Kovacic A, et al. Pan drug-resistant environmental isolate of *Acinetobacter baumannii* from Croatia. Microb Drug Resist 2017;23:494–6.

[144] Tafoukt R, Leangapichart T, Hadjadj L, et al. Characterisation of *bla*<sub>OXA-538</sub>, a new variant of *bla*<sub>OXA-48</sub>, in Shewanella xiamenensis isolated from river water in Algeria. J Glob Antimicrob Resist 2018;13:70–3.

[145] Higgins PG, Hrenovic J, Seifert H, et al. Characterization of *Acinetobacter baumannii* from water and sludge line of secondary wastewater treatment plant. Water Res 2018;140:261–7.

[146] Le Terrier C, Masseron A, Uwaezuoke NS, et al. Wide spread of carbapenem-producing bacterial isolates in a Nigerian environment. J Glob Antimicrob Resist 2020;21:321–3.

[147] Hrenovic J, Seruga Music M, Durn G, et al. Carbapenem-resistant *Acinetobacter baumannii* recovered from swine manure. Microb Drug Resist 2019;25:725–30.

[148] Zou H, Zhou Z, Xia H, et al. Characterization of chromosome-mediated *bla*<sub>OXA-894</sub> in Shewanella xiamenensis isolated from pig wastewater. Int J Environ Res Public Health 2019;16:3768.

[149] Qiao M, Ying GG, Singer AC, et al. Review of antibiotic resistance in China and its environment. Environ Int 2018;110:160–72.

[150] Subirats J, Royo E, Balcazar JL, et al. Real-time PCR assays for the detection and quantification of carbapenemase genes (*bla*<sub>KPC</sub>, *bla*<sub>NDM</sub>, and *bla*<sub>OXA-48</sub>) in environmental samples. Environ Sci Pollut Res Int 2017;24:6710–14.

[151] Nasri E, Subirats J, Sanchez-Melsio A, et al. Abundance of carbapenemase genes (*bla*<sub>KPC</sub>, *bla*<sub>NDM</sub> and *bla*<sub>OXA-48</sub>) in wastewater effluents from Tunisian hospitals. Environ Pollut 2017;229:371–4.

[152] Proia L, Anzil A, Borrego C, et al. Occurrence and persistence of carbapenemases genes in hospital and wastewater treatment plants and propagation in the receiving river. J Hazard Mater 2018;358:33–43.

[153] Marathe NP, Berglund F, Razavi M, et al. Sewage effluent from an Indian hospital harbors novel carbapenemases and integron-borne antibiotic resistance genes. Microbiome 2019;7:97.

[154] Lubbert C, Baars C, Dayakar A, et al. Environmental pollution with antimicrobial agents from bulk drug manufacturing industries in Hyderabad, South India, is associated with dissemination of extended-spectrum *β*-lactamase and carbapenemase-producing pathogens. Infection 2017;45:479–91.

[155] Dias MF, da Rocha Fernandes G, Cristina de Paiva M, et al. Exploring the resistance, virulence and microbiome of drinking water in environmental and clinical settings. Water Res 2020;174:115630.

[156] Tuo H, Yang Y, Tao X, et al. The prevalence of colistin resistant strains and antibiotic resistance gene profiles in Funan River. China. Front Microbiol 2018;9:3094.

[157] Bondarczkuk K, Piotrowska-Seget Z. Microbial diversity and antibiotic resistance in a final effluent-receiving lake. Sci Total Environ 2019;650:2951–61.

[158] Bartley PS, Dmitrovic TN, Moretto VT, et al. Antibiotic resistance in Enterobacteriaceae from surface waters in urban Brazil highlights the risks of poor sanitation. Am J Trop Med Hyg 2019;100:1369–77.

[159] Klase G, Lee S, Liang S, et al. The microbiome and antibiotic resistance in integrated fishfarm water: implications of environmental public health. Sci Total Environ 2019;649:1491–501.

[160] Yang F, Zhang K, Zhi S, et al. High prevalence and dissemination of *β*-lactamase genes in swine farms in Northern China. Sci Total Environ 2019;651:2507–13.

[161] Girlich D, Poirel L, Szczepanowski R, et al. Carbapenem-hydrolyzing GES-5-encoding gene on different plasmid types recovered from a bacterial community in a sewage treatment plant. Appl Environ Microbiol 2012;78:1292–5.

[162] Mi R, Patidar R, Farenhorst A, et al. Detection of fecal bacteria and antibiotic resistance genes in drinking water collected from three First Nations communities in Manitoba, Canada. FEMS Microbiol Lett 2019;366 fnz067.

[163] Dang B, Mao D, Luo Y. Complete nucleotide sequence of pGA45, a 140,698-bp IncFIIY plasmid encoding *bla*<sub>IMI-3</sub>-mediated carbapenem resistance, from river sediment. Front Microbiol 2016;7:188.

[164] Khan FA, Soderquist B, Jass J. Prevalence and diversity of antibiotic resistance genes in Swedish aquatic environments impacted by household and hospital wastewater. Front Microbiol 2019;10:688.

[165] Wang Y, Zhang R, Li J, et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nat Microbiol 2017;2:16260.

[166] Wang RN, Zhang Y, Cao ZH, et al. Occurrence of super antibiotic resistance genes in the downstream of the Yangtze River in China: prevalence and antibiotic resistance profiles. Sci Total Environ 2019;651:1946–57.

[167] Stange C, Yin D, Xu T, et al. Distribution of clinically relevant antibiotic resistance genes in Lake Tai. China. Sci Total Environ 2018;655:337–46.

[168] Zhou ZC, Feng WQ, Han Y, et al. Prevalence and transmission of antibiotic resistance and microbiota between humans and water environments. Environ Int 2018;121:1155–61.

[169] Rathinasabapathi P, Hiremath DS, Arunraj R, et al. Molecular detection of New Delhi metallo-*β*-lactamase-1 (NDM-1) positive bacteria from environmental and drinking water samples by loop mediated isothermal amplification of *bla*<sub>NDM-1</sub>. Indian J Microbiol 2015;55:400–5.

[170] Ahmad ZS, Sreekrishnan TR, Hands CL, et al. Increased waterborne *bla*<sub>NDM-1</sub> resistance gene abundances associated with seasonal human PIL-gimages to the Upper Ganges River. Environ Sci Technol 2014;48:3014–20.

[171] Gatica J, Tripathi V, Green S, et al. High throughput analysis of integron gene cassettes in wastewater environments. Environ Sci Technol 2016;50:11825–36.

[172] Fernando DM, Tun HM, Poole J, et al. Detection of antibiotic resistance genes in source and drinking water samples from a First Nations Community in Canada. Appl Environ Microbiol 2016;82:4676–75.

[173] Devarajan N, Laffite A, Graham ND, et al. Accumulation of clinically relevant antibiotic-resistance genes, bacterial load, and metals in freshwater lake sediments in Central Europe. Environ Sci Technol 2015;49:6528–37.

[174] Le TH, Ng C, Tran NH, et al. Removal of antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in municipal wastewater by membrane bioreactor systems. Water Res 2018;145:498–508.

[175] Makowska N, Philips A, Dabert M, et al. Metagenomic analysis of *β*-lactamase and carbapenemase genes in the wastewater resistome. Water Res 2020;170:115277.

[176] Voigt AM, Zacharias N, Timm C, et al. Association between antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in anthropogenic wastewater—an evaluation of clinical influences. Chemosphere 2020;241:125032.

[177] Tacao M, Silva I, Henriques I. Culture-independent methods reveal high diversity of OXA-48-like genes in water environments. J Water Health 2017;15:519–25.

[178] Bengtsson-Palme J, Hammaren R, Pal C, et al. Elucidating selection processes for antibiotic resistance in sewage treatment plants using metagenomics. Sci Total Environ 2016;572:697–712.

[179] Voigt AM, Ciorba P, Dohla M, et al. The investigation of antibiotic residues, antibiotic resistance genes and antibiotic-resistant organisms in a drinking water reservoir system in Germany. Int J Hyg Environ Health 2020;224:113449.

[180] Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006;60:561–88.

[181] Smith JM, Feil EJ, Smith NH. Population structure and evolutionary dynamics of pathogenic bacteria. Bioessays 2000;22:1115–22.

[182] Ribeiro-Gonçalves B, Francisco AP, Vaz C, et al. PHYLOViS Online: web-based tool for visualization, phylogenetic inference, analysis and sharing of minimum spanning trees. Nucleic Acids Res 2016;44:W246–51.

[183] Brink AJ. Epidemiology of carbapenem-resistant Gram-negative infections globally. Curr Opin Infect Dis 2019;32:609–16.

[184] Pitout JDD, Peirano G, Kock MM, et al. The global ascendency of OXA-48-type carbapenemases. Clin Microbiol Rev 2019;33:e00102–19.

[185] Uchida H, Tada T, Toha M, et al. Emergence in Japan of an isolate of *Klebsiella pneumoniae* co-harbouring *bla*<sub>KPC-2</sub> and rmtB. J Glob Antimicrob Resist 2019;17:157–9.

[186] Lu MC, Tang HL, Chiou CS, et al. Clonal dissemination of carbapenemase-producing *Klebsiella pneumoniae*: two distinct sub-lineages of sequence type 11 carrying *bla*<sub>KPC-2</sub> and *bla*<sub>OXA-48</sub>. Int J Antimicrob Agents 2018;52:658–62.

[187] Fu P, Tang Y, Li G, et al. Pandemic spread of *bla*<sub>KPC-2</sub> among *Klebsiella pneumoniae* ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids. Int J Antimicrob Agents 2019;54:117–24.

[188] Jelic M, Butic I, Plecko V, et al. KPC-producing *Klebsiella pneumoniae* isolates in Croatia: a nationwide survey. Microb Drug Resist 2016;22:662–7.

[189] García-Fernández A, Villa L, Carta C, et al. *Klebsiella pneumoniae* ST258 producing KPC-3 identified in Italy carries novel plasmids and Ompk36/Ompk35 porin variants. Antimicrob Agents Chemother 2012;56:2143–5.

[190] Loconsole D, Accogli M, De Robertis AL, et al. Emerging high-risk ST101 and ST307 carbapenem-resistant *Klebsiella pneumoniae* clones from bloodstream infections in Southern Italy. Ann Clin Microbiol Antimicrob 2020;19:24.

[191] Pereira PS, de Araujo CF, Seki LM, et al. Update of the molecular epidemiology of KPC-2-producing *Klebsiella pneumoniae* in Brazil: spread of clonal complex 11 (ST11, ST437 and ST340). J Antimicrob Chemother 2013;68:312–16.

[192] Zhu J, Sun L, Ding B, et al. Outbreak of NDM-1-producing *Klebsiella pneumoniae* ST76 and ST37 isolates in neonates. Eur J Clin Microbiol Infect Dis 2016;35:611–18.

[193] Bonura C, Giuffrè M, Aleo A, et al. An update of the evolving epidemic of *bla*<sub>KPC</sub> carrying *Klebsiella pneumoniae* in Sicily, Italy, 2014: emergence of multiple non-ST258 clones. PLoS One 2015;10:e0132936.

[194] Khan ER, Aung MS, Paul SK, et al. Prevalence and molecular epidemiology of clinical isolates of *Escherichia coli* and *Klebsiella pneumoniae* harboring extended-spectrum *β*-lactamase and carbapenemase genes in Bangladesh. Microb Drug Resist 2018;24:1568–79.

[195] Flerlage T, Brazelton de Cardenas JN, Garner CD, et al. Multiple NDM-5-expressing *Escherichia coli* isolates from an immunocompromised pediatric host. Open Forum Infect Dis 2020;7 ofaa018.

[196] Dagher C, Salloum T, Alousi S, et al. Molecular characterization of carbapenem resistant *Escherichia coli* recovered from a tertiary hospital in Lebanon. PLoS One 2018;13:e0203323.

[197] Falgenhauer L, Nordmann P, Imirzalioglu C, et al. Cross-border emergence of clonal lineages of ST38 *Escherichia coli* producing the OXA-48-like carbapenemase OXA-244 in Germany and Switzerland. Int J Antimicrob Agents 2020;56:106157.

[198] Qin S, Zhou M, Zhang Q, et al. First identification of NDM-4-producing *Escherichia coli* ST410 in China. Emerg Microbes Infect 2016;5:e118.

[199] Yagoubat M, Ould El-Hadj-Khelil A, Malki A, et al. Genetic characterisation of carbapenem-resistant Gram-negative bacteria isolated from the University Hospital Mohamed Boudiaf in Ouarla, southern Algeria. J Glob Antimicrob Resist 2017;8:55–9.

[200] Sanderson H, Fricker C, Brown RS, et al. Antibiotic resistance genes as an emerging environmental contaminant. Environ Rev 2016;24:205–18.