Carbapenem Resistance among Marine Bacteria—An Emerging Threat to the Global Health Sector

The emergence of antibiotic resistance among pathogenic microorganisms is a major issue for global public health, as it results in acute or chronic infections, debilitating diseases, and mortality. Of particular concern is the rapid and common spread of carbapenem resistance in healthcare settings. Carbapenems are a class of critical antibiotics reserved for treatment against multidrug-resistant microorganisms, and resistance to this antibiotic may result in limited treatment against infections. In addition to in clinical facilities, carbapenem resistance has also been identified in aquatic niches, including marine environments. Various carbapenem-resistant genes (CRGs) have been detected in different marine settings, with the majority of the genes incorporated in mobile genetic elements, i.e., transposons or plasmids, which may contribute to efficient genetic transfer. This review highlights the potential of the marine environment as a reservoir for carbapenem resistance and provides a general overview of CRG transmission among marine microbes.


Introduction
Antibiotic resistance (AR) is a public health crisis that leads to increasingly complex and expensive treatments, longer hospital stays, and higher mortality [1]. The Centre for Disease Prevention and Control (CDC) has estimated that in the United States alone, two million people are infected by antibiotic-resistant bacteria (ARB), resulting in 23,000 fatalities every year [2]. The WHO estimates that this number will increase to approximately 10 million deaths per year by 2050 if the AR problem is not addressed [3,4]. ARB can be found in humans, animals, plants, and the environment, such as in water and soil [5], and may spread among humans, between humans and animals, from humans or animals to the environment, or vice versa [6][7][8][9][10]. The factors contributing to the emergence and spread of ARB include the overuse or misuse of antimicrobials; poor infection prevention and control in healthcare facilities; lack of access to clean water, sanitation, and hygiene for humans and animals; lack of knowledge and awareness; and lack of legislation enforcement [5]. Furthermore, the rapid global spread of ARB that have acquired new resistance mechanisms has, alarmingly, resulted in multi-and pan-resistant bacteria, also known as "superbugs" [5]. Consequently, only a few antibiotics are left to treat human infections caused by these multi-drug-resistant bacteria [11][12][13]. The WHO, therefore, suggests that prevention and containment methods are required to decrease the spread of ARB, and these include improved methods for antibiotic prescription, the regulation of antibiotics use, the development of new antimicrobial drugs and vaccines, and improved surveillance of ARB [5].
carbapenemase-producing Serratia fonticola was reported in drinking water in Portugal [69], and CPE including E. coli, Kluyvera, Providencia, Klebsiella, and Citrobacter species, and non-fermenting Gram-negative species, such as Shewanella spp., Pseudomonas spp., and Acinetobacter spp., were found to contain blaOXA-48-type carbapenemase in USA drinking waters [66]. In addition, non-fermenting Gram-negative rods carrying the bla NDM gene were isolated from New Delhi drinking water [67]. Thus, the aquatic environments may serve as a vehicle by which CRB or CRGs could be disseminated from one aquatic ecosystem to another (see Figure 1). Shigella boydii, Aeromonas caviae, and Vibrio cholerae from seepage and drinking water samples [67]. Multi-resistant bacteria and ARGs were detected at a drinking water intake at Lake Geneva, Switzerland, which was 3.2 km away from a WWTP outlet [68]. Furthermore, CRB have also been found in drinking water in several parts of the world. For instance, carbapenemase-producing Serratia fonticola was reported in drinking water in Portugal [69], and CPE including E. coli, Kluyvera, Providencia, Klebsiella, and Citrobacter species, and non-fermenting Gram-negative species, such as Shewanella spp., Pseudomonas spp., and Acinetobacter spp., were found to contain blaOXA-48-type carbapenemase in USA drinking waters [66]. In addition, non-fermenting Gram-negative rods carrying the blaNDM gene were isolated from New Delhi drinking water [67]. Thus, the aquatic environments may serve as a vehicle by which CRB or CRGs could be disseminated from one aquatic ecosystem to another (see Figure 1). The dense bacterial communities in WWTPs can also facilitate genetic exchange between bacteria, which can lead to the horizontal transfer of resistance genes between clinical pathogens and environmental microorganisms or vice versa [7,70]. For example, Citrobacter freundii and Enterobacter cloacae detected in hospital sewage have been found to contain the same blaKPC-2 gene [71]. The species Pseudomonas monteilii, Brevundimonas diminuta, and Enterobacter ludwigii have been found to contain the same genetic variant of the blaVIM-13 gene in sewage [22]. Finding identical CRGs in different bacterial taxa indicates that sewage is a suitable environment for horizontal resistance gene transfer.

Distribution of Carbapenem Resistance in Marine Systems
The dissemination of AR in seawater may be influenced by discharges from coastal runoff, aquaculture, polluted rivers, and WWTP effluents, which frequently contain resistant bacteria and resistant genes [72][73][74][75][76][77]. A study described that CRB communities in seawater and storm water samples did not differ significantly at the investigated sites, and the phylogenetic analysis showed that their CR isolates often belonged to the same species [25]. CPE, such as Enterobacter spp. and E. coli carrying blaIMI-2, were also isolated from river estuaries and beach water. Further molecular analysis and genome The dense bacterial communities in WWTPs can also facilitate genetic exchange between bacteria, which can lead to the horizontal transfer of resistance genes between clinical pathogens and environmental microorganisms or vice versa [7,70]. For example, Citrobacter freundii and Enterobacter cloacae detected in hospital sewage have been found to contain the same bla KPC-2 gene [71]. The species Pseudomonas monteilii, Brevundimonas diminuta, and Enterobacter ludwigii have been found to contain the same genetic variant of the bla VIM-13 gene in sewage [22]. Finding identical CRGs in different bacterial taxa indicates that sewage is a suitable environment for horizontal resistance gene transfer.

Distribution of Carbapenem Resistance in Marine Systems
The dissemination of AR in seawater may be influenced by discharges from coastal runoff, aquaculture, polluted rivers, and WWTP effluents, which frequently contain resistant bacteria and resistant genes [72][73][74][75][76][77]. A study described that CRB communities in seawater and storm water samples did not differ significantly at the investigated sites, and the phylogenetic analysis showed that their CR isolates often belonged to the same species [25]. CPE, such as Enterobacter spp. and E. coli carrying bla IMI-2 , were also isolated from river estuaries and beach water. Further molecular analysis and genome comparisons revealed the high similarity of these riverine and marine CRB from samples that were collected one month apart [77]. Thus, local sources, such as stormwaters and rivers, are an important source of CRB in the seawater and may have a significant effect on the composition of CRB in the marine environment (see Figure 1).

Potential for CR Transfer and Reservoir in the Marine Environment
The marine environment may contribute to the further dissemination of CRGs between different bacterial strains by providing a medium in which horizontal gene transfer can take place [98,99]. For example, a study of CR in the coastal environment found that most of the CR aquatic isolates were assigned to the genus Pseudomonas, including the species P. asplenii, P. monteilii, P. fulva, P. plecoglossicida, P. stutzeri, P. taiwanensis, and P. xanthomarina. In the genus Pseudomonas (family Pseudomonadaceae), the production of carbapenemases, such as IMP, VIM, NDM, and KPC, is considered to be the predominant mechanism underlying CR [9,18,100,101]. The CRGs are frequently located in the mobile genetic elements, which facilitate their horizontal transfer between different species [102][103][104][105]. Thus, horizontal gene transfer might have been involved in the dissemination of CRGs in the Pseudomonas species, similar to what has been observed in the family Enterobacteriaceae [40,41,106,107].
Several CRGs, such as bla KPC , bla NDM , bla GES , and bla OXA48-like , have been found in different members of Enterobacteriaceae, including K. pneumoniae, Aeromonas punctata, A. hydrophila, E. coli, E. cloacae, E. kobei, and E. asburiae, in recreational seawaters [91,93]. Furthermore, E. coli from terrestrial sources could transfer its bla CTX-M-15 gene to Pantoea agglomerans and Raoultella terrigena in the coastal seawater [108]. Another study reported that in seawater samples, Rheinheimera spp., which are typical marine bacteria [109,110], have been found to share an identical MBL gene utilizing plasmid transfer and chromosomal integration with the species C. freundii and E. cloacae, which are not typically found in seawater [25]. In addition, Variovorax spp. carried the NDM-type genes, which were likely acquired from the species E. coli, K. pneumoniae, and A. baumannii. These utilized plasmids have also been reported in this coastal water study [25].
Marine samples have also been found to house CRB from genera such as Chromobacterium, Rheinheimera, Variovorax, Aquiflexum, Chitinophaga, Herbaspirillum, and Xanthobacter, which have not previously been known to have CR, indicating the potential for new resistance genes [25]. As marine environments are not usually exposed to high concentrations of clinically relevant carbapenems or other β-lactam antibiotics [111,112], marine bacteria may evolve novel CR due to the selection pressure of natural β-lactam antibiotics produced by other marine microorganisms [113,114]. This could, for example, include aureoverticillactam and lajollamycin found in the marine species Streptomyces aureoverticillatus [115] and S. nodosus [116], respectively (see Figure 1). As such, several novel carbapenemases have been recently discovered. These include ElBla2 MBL from the species Erythrobacter litoralis (family Sphingomonadaceae), which has an amino acid sequence similarity to NDM-1 [78]; the PH-1 MBL gene from the species Pelagibacterium halotolerans (family Hyphomicrobiaceae) [80]; and the RH-B3-MBL gene from the genus Rheinheimera (family Chromatiaceae) [25]. These findings provide further evidence that the marine environment represents an unexplored reservoir of novel carbapenemases.
In addition, there is growing evidence that CRGs have been carried by human opportunistic pathogens into seawaters [73,80,94]. For example, CRGs, such as KPC, IMP, VIM, SPM, NDM, and OXA-carbapenemases, have been identified in E. coli, K. pneumoniae, Acinetobacter spp., Aeromonas spp., Enterobacter spp., and Pseudomonas spp. in recreational coastal waters of Rio de Janeiro [73,76], Israel [77], and Ireland [95]. This prevalence has public health implications [26,86], since coastal waters are frequently used for recreational and sport purposes. CR infections can, in such circumstances, be acquired through common routes of pathogen exposure to humans, including ingestion, inhalation, and dermal or eye contact with the beach waters [128,129] (see Figure 2). For example, the risk of experiencing symptoms of gastrointestinal, ear, and skin infections from exposure to seawater with bacterial contamination is higher in bathers than in non-bathers [26,129]. Open wounds in soft tissues are also vulnerable to infection by opportunistic human pathogens. For example, a soft tissue infection by Microbacterium lacus was acquired after a bather's elbow was bumped on a rock while swimming [124]. Considering the spread of CRB in marine environments that are frequently used for human activities, the potential risk of CRB transfer from this reservoir to humans is still great. Further in-depth studies to quantify the risk of human infection with these CRB in recreational seawater should be performed.
VIM, SPM, NDM, and OXA-carbapenemases, have been identified in E. moniae, Acinetobacter spp., Aeromonas spp., Enterobacter spp., and Pseudomon reational coastal waters of Rio de Janeiro [73,76], Israel [77], and Ireland [95 This prevalence has public health implications [26,86], since coastal w quently used for recreational and sport purposes. CR infections can, in stances, be acquired through common routes of pathogen exposure to huma ingestion, inhalation, and dermal or eye contact with the beach waters [128 ure 2). For example, the risk of experiencing symptoms of gastrointestinal, infections from exposure to seawater with bacterial contamination is high than in non-bathers [26,129]. Open wounds in soft tissues are also vulnerab by opportunistic human pathogens. For example, a soft tissue infection by M lacus was acquired after a bather's elbow was bumped on a rock while swi Considering the spread of CRB in marine environments that are frequently man activities, the potential risk of CRB transfer from this reservoir to h great. Further in-depth studies to quantify the risk of human infection with recreational seawater should be performed. Figure 2. Schematic diagram showing the route of potential CRB transmission from vironment to humans and the potential health problems that may occur. The practice activities, such as sport and fishing, in CR-contaminated coastal environments may tial human illnesses, such as dermal or eye problems and gastrointestinal infection routes of transmission include accidentally ingested or inhaled beach waters that ar by CR. Figure 2. Schematic diagram showing the route of potential CRB transmission from the coastal environment to humans and the potential health problems that may occur. The practice of recreational activities, such as sport and fishing, in CR-contaminated coastal environments may result in potential human illnesses, such as dermal or eye problems and gastrointestinal infections. The possible routes of transmission include accidentally ingested or inhaled beach waters that are contaminated by CR.

Conclusions
The global spread of CR is becoming a major threat to public health and has worsened with the detection of resistance in last-line antibiotics, including carbapenems [1, 15,16]. CR has not only been reported in pathogenic bacteria [6,9,10,31,130], but also in environmentally derived bacteria [86,108,131,132]. As non-clinical environments, such as sewage, rivers, lakes, and oceans, may serve as the media for the transmission of CR [24,73,76,133,134], studies of the further dissemination of CR in these matrices are critically important. In the coastal environment, CRB and CRGs seem to be transported from terrestrial sources through stormwater runoff, wastewater discharges, and polluted rivers [21,25,42,57], which had a significant effect on the diversity and CRB load in the marine environment.
To date, a large variety of CRGs have been identified, and the transfer of CRGs between different bacteria has been characterized [22,49,135]. Most CRGs are located on mobile genetic elements, such as plasmids or transposons, and their mobility contributes to the rapid spread of CRGs between bacteria [16,136]. The identification of CRGs, including novel carbapenemases in non-targeted marine bacteria [25,78,80], suggests either that CRGs have been horizontally transferred from non-marine bacteria (e.g., fecal contaminant) to marine bacteria or that novel CRGs have evolved in marine lineages due to continuing selection by naturally produced β-lactam antibiotics in the marine environment.
Given that the CRB identified in coastal environments are relevant to human health, public health concerns may arise due to the fact that beach water is mostly designated for recreational and sporting activities [24,26,73,137]. Future investigation of CR in the coastal environment is necessary to understand the resistant epidemiology and quantify the potential risk for CRB to cause human illnesses.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.