Antimicrobial Resistance in Romania: Updates on Gram-Negative ESCAPE Pathogens in the Clinical, Veterinary, and Aquatic Sectors

Multidrug-resistant Gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, and members of the Enterobacterales order are a challenging multi-sectorial and global threat, being listed by the WHO in the priority list of pathogens requiring the urgent discovery and development of therapeutic strategies. We present here an overview of the antibiotic resistance profiles and epidemiology of Gram-negative pathogens listed in the ESCAPE group circulating in Romania. The review starts with a discussion of the mechanisms and clinical significance of Gram-negative bacteria, the most frequent genetic determinants of resistance, and then summarizes and discusses the epidemiological studies reported for A. baumannii, P. aeruginosa, and Enterobacterales-resistant strains circulating in Romania, both in hospital and veterinary settings and mirrored in the aquatic environment. The Romanian landscape of Gram-negative pathogens included in the ESCAPE list reveals that all significant, clinically relevant, globally spread antibiotic resistance genes and carrying platforms are well established in different geographical areas of Romania and have already been disseminated beyond clinical settings.


Introduction
Gram-negative bacteria represent one of the most significant pathogens involved in public health issues. Among them, members of the families Enterobacterales, Moraxellales, and Pseudomonadales have a major clinical significance, being key Gram-negative pathogens listed in the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) or later designated ESCAPE group (E. faecium, S. aureus, Clostridioides difficile, A. baumannii, P. aeruginosa, Enterobacterales), as well as in the WHO priority pathogens list for R&D of new antibiotics in Priority 1: CRITICAL list (namely carbapenem-resistant A. baumannii, carbapenem-resistant P. aeruginosa, and carbapenem-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacterales) [1]. The Centers for Disease Control and Prevention (CDC) described the dramatic increase of antibiotic resistance (AR) during the last decades as one of the most critical threats to public health, with very few effective antimicrobials left, either novel or old molecules [2,3], therefore requiring concerted research and management efforts [4].

Aminoglycoside Resistance
Aminoglycoside resistance in Gram-negative bacilli is mainly encoded by enzymatic resistance mechanisms (by the production of aminoglycoside-modifying enzymes-AMEs) but also by alteration of the ribosome structure (by 16S rRNA methyltransferases) and limited antibiotic uptake (due to the loss of cell membrane permeability or hyperactivity of the efflux pumps) [36][37][38]. AMEs are classified as aminoglycoside phosphotransferases (APH), aminoglycoside acetyltransferases (AAC), aminoglycoside nucleotidyltransferases (ANT), and aminoglycoside adenylyltransferases (AAD) [36]. The most frequently detected AME-encoding genes in Gram-negative strains listed in the ESCAPE group are presented in Table 2.

Fosfomycin Resistance
Fosfomycin is a broad-spectrum antimicrobial agent that inhibits the final step of peptidoglycan biosynthesis. Resistance is attributed to the modification of transporters across the cytoplasmic membrane, amino acid substitution in the MurA active site, which decreases fosfomycin binding affinity, and the production of the fosfomycin-inactivating enzyme FosA [224].

Antifolate Resistance
Antifolate antibiotics inhibit purine metabolism and DNA and RNA synthesis by interfering with folic acid biosynthesis. Sulfonamides bind dihydropteroate synthase (DHPS), a catalytic enzyme in the folic acid biosynthesis pathway, inhibiting dihydrofolic acid formation [225]. Trimethoprim is a dihydrofolate reductase (DHFR) inhibitor. Sulfonamides combined with trimethoprim (such as sulfamethoxazole) are well-known folate inhibitors. Resistance to sulfonamides occurs due to mutations of the folP gene encoding DHPS or the acquisition of alternative DHPS genes (sul1, sul2, sul3, sul4) with low affinity to sulfonamides [226]. Trimethoprim resistance is mediated by dfr genes encoding trimethoprim-resistant dihydrofolate reductases.
Gram-negative pathogens have developed resistance mechanisms to all antibiotics used for therapy ( Figure 1).

Mobile Genetic Elements (MGEs)
MGEs [insertion sequences (IS), transposons (Tn), integrons, resistance islands, and plasmids] play a significant role in the evolution of prokaryotic genomes, conferring adaptative traits, including AR. Thus, MGEs can retain, capture, and disseminate the ARGs between bacterial strains or species responsible for the emergence of MDR [227,228]. encoding DHPS or the acquisition of alternative DHPS genes (sul1, sul2, sul3, sul4) with low affinity to sulfonamides [226]. Trimethoprim resistance is mediated by dfr genes encoding trimethoprim-resistant dihydrofolate reductases.
Gram-negative pathogens have developed resistance mechanisms to all antibiotics used for therapy ( Figure 1).

Mobile Genetic Elements (MGEs)
MGEs [insertion sequences (IS), transposons (Tn), integrons, resistance islands, and plasmids] play a significant role in the evolution of prokaryotic genomes, conferring adaptative traits, including AR. Thus, MGEs can retain, capture, and disseminate the ARGs between bacterial strains or species responsible for the emergence of MDR [227,228].

Insertion Sequences (IS)
IS are MGE that carry one or two transposase (tnp) genes and are responsible for the intracellular transportation of ARGs. ISs can be found across all prokaryotes, and their role in AR dissemination was well documented, not only by being able to mobilize ARGs but also by activating or inactivating specific genes in the bacterial chromosome, acting as promoters for silent ARGs or enhancing their expression, or, as in the case of certain enterobacteria, by inactivating certain porin-encoding genes or regulator genes [229,230]. In addition, two ISs can also form a composite transposon and sequester the genes between them [231].

Insertion Sequences (IS)
IS are MGE that carry one or two transposase (tnp) genes and are responsible for the intracellular transportation of ARGs. ISs can be found across all prokaryotes, and their role in AR dissemination was well documented, not only by being able to mobilize ARGs but also by activating or inactivating specific genes in the bacterial chromosome, acting as promoters for silent ARGs or enhancing their expression, or, as in the case of certain enterobacteria, by inactivating certain porin-encoding genes or regulator genes [229,230]. In addition, two ISs can also form a composite transposon and sequester the genes between them [231].
The notorious IS26 (or IS6) family is a well-known example of IS capable of sequestering and mobilizing ARGs. Members of the IS family are often found in arrays, in direct and/or inverted repeats, in MDR plasmids described in Gram-negative ESCAPE strains, and are able to capture virtually every ARG [251].

Clinical Significance of Antibiotic-Resistant Gram-Negative Pathogens
The MDR and virulence potential of A. baumannii are responsible for hospital and community-acquired infections [274]. A. baumannii is recognized as an opportunistic nosocomial pathogen, mainly in immunocompromised patients, and is frequently associated with therapeutic failures, especially during the COVID-19 pandemic. Several countries have reported that COVID-19 was associated with secondary MDR carbapenem-resistant A. baumannii (MDR CRAB) infections of the lower respiratory tract in intensive care unit (ICU) patients, emphasizing the importance of limiting the risk of co-infection and the dissemination of MDR CRAB strains in ICUs [275][276][277].
Carbapenem-resistant P. aeruginosa (CR-PA) is a major healthcare-associated pathogen worldwide [278]. P. aeruginosa is the primary cause of ventilator-associated pneumonia (VAP) in long-term acute care hospitals and hospital wards and the second most common cause of VAP in intensive care units. It is also the third most common cause of catheterrelated urinary tract infections [279]. In P. aeruginosa, several mechanisms are responsible for carbapenem resistance. The first mechanism is the efflux pump, which is mediated by overexpression of the MexAB-OprM efflux pump, resulting in resistance to most β-lactam drugs except for imipenem. The second mechanism is the overproduction of AmpC betalactamase and the inactivation of the OprD outer membrane protein. This combination can lead to resistance to essentially all antipseudomonal β-lactams. Another resistance mechanism is the production of carbapenemases [280,281], which significantly alter the efficacy of commonly used antipseudomonal agents, including ceftazidime, cefepime, and piperacillin-tazobactam, as well as the newly introduced β-lactam/β-lactamase inhibitor combinations such as ceftolozane-tazobactam, imipenem-relebactam, and ceftazidimeavibactam. The carbapenem resistance determinants carried by P. aeruginosa are often encoded on plasmids, such as IncP type; class I integrons, for example, those carrying the bla VIM gene; and other MGE, such as those associated with insertion sequence common region (ISCR) elements [114]. In addition, these isolates frequently carry additional resistance determinants to fluoroquinolones and aminoglycosides. Carbapenemase-producing P. aeruginosa (CP-PA) is often resistant to these therapeutic options, thus making treatment failure likely. CP-PA has also been associated with nosocomial spread, prompting infection prevention interventions [280].
The Enterobacterales order, as defined by Adelou et al. in 2016, comprises Gramnegative, non-spore-forming, rod-shaped, and facultative anaerobes bacteria. The order contains the families Enterobacteriaceae, Erwiniaceae, Pectobacteriaceae, Yersiniaceae, Hafniaceae, Morganellaceae, and Budviciaceae, some of which are members of the normal microbiota of the mammalian gastrointestinal tract [282]. The drastic rise in the incidence of MDR and extended drug-resistant (XDR) pathogens belonging to the Enterobacteriaceae group is a significant economic problem as these pathogens are prevalent natural residents of the human and animal microbiomes and spread quickly between humans. Moreover, Enterobacterales easily acquire ARGs via MGEs [283].
Most notable in Enterobacterales is the resistance to β-lactam antibiotics due to ESBL production, mainly in E. coli and K. pneumoniae, followed by aminoglycoside and fluoroquinolone resistance (Tables 1 and 2). These resistance phenotypes are often coupled, leading to MDR and the necessity to use last-resort antibiotics [284].
K. pneumoniae is the causative agent of about one-third of all Gram-negative infections (urinary tract infections, cystitis, pneumonia, surgical wound infections, endocarditis, septicemia, necrotizing pneumonia, pyogenic liver abscesses, and endogenous endophthalmitis), associated with high mortality rates and extended hospitalization, coupled with high economic costs. Due to selective pressure caused by antibiotic usage, K. pneumoniae collects ARGs, which led to the development of XDR strains harboring a 'super resistome'. These include the emergence of hypervirulent K. pneumoniae (hvKp) or hypermucoviscous K. pneumoniae (HMKP), usually susceptible to last-line antibiotics (carbapenems and colistin) [285,286]. The continuous global dissemination of high-risk MDR and XDR K. pneumoniae highlights their complex evolution, involving the transfer and spread of ARGs and epidemic plasmids [287,288]. Most of the carbapenemase and/or ESBL-producing K. pneumoniae strains, as well as those harboring aminoglycoside resistance, belong to specific clones CC (clonal complex) 258, CC15, and CC14 [289], while colistin-resistant clones mainly belong to CC11, 43, and 258 [290][291][292][293].
Enterobacter spp. are increasingly described as contributing to the dissemination of infections caused by carbapenem resistant strains. Amongst the 22 species of this genus, Enterobacter aerogenes, E. cloacae, and E. hormaechei are the most frequently isolated species in clinical infections, mainly in immunocompromised patients and those hospitalized in ICU, due to their adaptation to the hospital environment and their ability to efficiently acquire numerous genetic mobile elements containing resistance and virulence genes [294].
E. coli is particularly interesting since it represents a significant part of the normal microbiota, but it can also cause severe infections in humans and animals. In humans, E. coli can cause infections in practically every anatomical site of the human body, involving urinary tract infections, appendicitis, pneumonia, the bloodstream, gastrointestinal infections, skin abscesses, intra-amniotic and puerperal infections in pregnant women, meningitis, and endocarditis. Moreover, E. coli is involved in community-acquired and healthcare-related infections and can cause disease in all age groups [296,297].
E. coli is the second bacteria (after Klebsiella) involved in human infections associated with MDR bacterial infections. Furthermore, the significant increase in the emergence and dissemination of E. coli to the main antibiotic classes (β-lactams, quinolones, aminoglycosides, sulfonamides, and fosfomycin), including the last-resort carbapenems and polymyxins, is correlated with prolonged hospital stays and patient deaths [297,298].

Other Enterobacterales
Citrobacter spp., mainly C. freundii, are inhabitants of the intestinal tract and have been associated with nosocomial infections involving the urinary tract, liver, biliary tract, peritoneum, intestines, bone, respiratory tract, endocardium, wounds, soft tissue, meninges, and the bloodstream. The emergence of MDR Citrobacter strains is an increasing concern due to the production of AmpC, broad-spectrum β-lactamase, ESBL, or even carbapenemase, particularly MBL or KPC types. In addition, quinolone resistance (qnr and aac(6 )-Ib-cr genes), numerous qnrB alleles, and about 40 qnrB variants (located on the chromosome of Citrobacter spp., especially C. freundii) were described [299].
Hafnia alvei is rarely isolated from human samples. When it does, it is responsible for nosocomial infections, including gastroenteritis, urinary tract infections, meningitis, pneumonia, wound infections, soft tissue infections, endophthalmitis, and septicemia. The organism resides in the gastrointestinal tract of humans and many animals. Most infections with H. alvei are identified in patients with severe underlying diseases (e.g., malignancies) or after surgery or trauma. Besides its natural resistance to colistin and expression of AmpC chromosomal β-lactamase, it was described as the emergence of a Hafnia paralvei resistant to carbapenems due to a defective porin [300].
Morganella morganii is ubiquitous and is often associated with stool specimens collected from patients with symptoms of diarrhea. They are normal inhabitants of the gastrointestinal tract. M. morganii has intrinsic resistance to oxacillin, ampicillin, amoxicillin, and most first-and second-generation cephalosporins, macrolides, lincosamides, glycopeptides, fosfomycin, fusidic acid, and colistin. AR in M. morganii has been raised in recent years, mainly due to MGEs, leading to MDR and XDR strains [301].
Providencia spp. are usually isolated from patients with urinary tract infections and diarrhea and are associated with nosocomial outbreaks. Most commonly, P. rettgeri and P. stuartii represent the majority of MDR strains isolated and are intrinsically resistant to penicillins and the first-generation cephalosporins, aminoglycosides, tetracyclines (including tigecycline), and colistin [302,303].
Serratia spp., most commonly S. marcescens, is involved in nosocomial outbreaks and the colonization of diverse healthcare settings. S. marcescens has been associated with meningitis, sepsis, UTIs, skin infections, bloodstream infections, and respiratory infections. The intrinsic resistance to ampicillin, first-and second-generation cephalosporins, macrolides, and antimicrobial peptides, including colistin, is very challenging for clinical management. Moreover, some strains express the SME-1 enzyme, conferring resistance to imipenem, aztreonam, cephalosporins, and penicillins [304,305].
Salmonellae are Gram-negative bacteria that are pathogenic to humans and are traditionally subdivided into two major groups based on their clinical presentation: typhoidal Salmonella and non-typhoidal Salmonella. Typhoidal Salmonella, comprising the S. enterica subspecies enterica (hereafter Salmonella) serovars Typhi and Paratyphi A, B, and C, cause a systemic disease also known as enteric fever [306]. Human-restricted S. Typhi is the dominant cause of typhoid fever, with an estimated number of cases between 21.7 million and 26.9 million per year [307] and an estimated 217,000 deaths per year [308]. S. enterica constitutes a significant public health concern, and it is estimated to cause more than 300,000 annual deaths, mostly in developing countries [309]. This species is classified into hundreds of serovars based on surface antigenic composition. Some serovars (e.g., S. Typhi and S. Paratyphi) are host-adapted to humans, where they cause a systemic infection known as typhoid or paratyphoid fever and are therefore referred to as "typhoidal" serovars. Other serovars, such as S. Typhimurium, have a broad host range and, in humans, most often cause self-limiting gastroenteritis and are referred to as "non-typhoidal" serovars [310].
In Salmonella spp., particularly S. Typhi, antimicrobial resistance could be mediated by plasmid or chromosomal DNA. Usually, resistance is developed by the inactivation of antibacterial agents, alteration of drug targets, and employing various efflux pumps. In addition, external resistance factors may be actively mediated by gene transfer using virulence plasmids, phages, and MGEs [311]. S. Typhi typically has plasmids that contain several virulence factors and ARGs. These plasmids vary in size (50-90 kb) and carry the spv operon, which is significantly involved in causing infection. The genes of this operon are reportedly pivotal for bacterial proliferation in host cells and supposedly enhance the virulence of the pathogen [312]. Considering that most virulence plasmids are not selftransferable, some contain transgenes that enable the transfer of plasmids via conjugation. Incompatible (Inc) plasmids encode multiple antimicrobial resistance genes in S. Typhi and are classified into IncH1, IncH2, and IncH3. In addition, plasmids R27, pHCM1, and pAKU1 comprise a composite transposon that can harbor multidrug resistance in MDR S. Typhi strains [313].
Regarding the production of β-lactamases, TEM, SHV, and CTX-M are the main types of ESBLs in Salmonella spp., conferring resistance to penicillin and cephalosporin [314]. In S. Typhi, the presence of these genes has been attributed to the genetic transfer of resistance genes from other Gram-negative bacterial species [315]. In Salmonella spp., there were described genes encoding resistance to tetracycline (tetA, tetB, tetG), quinolones (qnrA, qnrB, qnrC, qnrS), and chloramphenicol (cat1 and cat2). Genetic elements identifying the mobile gene cassettes that carry multidrug-resistant genes are known as integrons. In S. Typhi, the presence of integrons (classes 1 and 2) equalizes the distribution of antimicrobial resistance, in which class 1 is more dominant [316].

Antibiotic Resistance in Romanian Hospital Settings
Romania is one of the European countries with the highest rates of MDR in A. baumannii clinical isolates (in 2020, the highest resistance percentages were recorded for fluoroquinolones, carbapenems, and aminoglycosides) [317]. Concerning P. aeruginosa, the highest resistance levels in 2020 were recorded for fluoroquinolones, carbapenems, and ceftazidime [318]. To the best of our knowledge, available data on molecular characterization of the non-fermenting strains is sourced from the major geographical areas of the country, including the capital city (Table 3).  Clinical A. baumannii strains from different Romanian regions exhibited CHLD-producing bla OXA-23 (West, North, Central, and South regions), followed by bla OXA-24/72 gene (North, Central, and South regions), and bla OXA-58 gene revealed by one study from the capital city, and respectively MBLs encoding bla VIM-2 and bla IMP-1 genes (South and North). The distribution of CHLDs and MBLs by isolation sources and period highlighted that carbapenem-encoding genes were not correlated over the period with the specific isolation sources, being described in strains isolated from infection sites or anal carriage. Studies from the West, Central, and South regions reported ESBL-encoding genes (bla PER-1 ; bla TEM-1 ; bla TEM-12 ; bla TEM-84 and bla SHV-12 ) from sterile or non-sterile isolation sources (Table 3). However, a limited number of studies have focused on the MGE carrying the CHLDs and revealed the presence of class 1 integrons, insertion sequences, transposons, or different plasmid types in A. baumannii recovered from South and Western Romanian intrahospital infections. A. baumannii reported from western and southern Romania belonged to high-risk international clones like ST2, ST1, ST636, and ST492.
Between 2008 and 2015, the most common carbapenemase-encoding genes in P. aeruginosa clinical strains isolated from intra-hospital infections or carriages in North, Central, and South Romania were bla VIM-2 , bla VIM-4 and bla IMP-13 . In addition, ESBL-encoding genes (bla SHVlike ; bla GESlike ; bla VEBlike ; bla TEMlike ) were encountered in P. aeruginosa strains isolated in the southern region of Romania.
Clinical enterobacterial isolates harbored mainly the ESBL bla CTX-M(-15) gene, followed by bla SHV and bla TEM (for which the ESBL phenotype depends on the gene variant). bla OXA-48 was the most commonly reported carbapenemase, followed by bla NDM and bla KPC (Table 4). Aminoglycoside resistance was mainly associated with AMEs, and all variants of PMQR have been described (Table 5).   Several studies investigated the mobile genetic platforms carrying the respective ARGs, particularly in the southern part of the country, and highlighted the presence of plasmids and integrons previously associated with AR without being associated with specific isolation sources. In addition, strains isolated from different infection sites of inpatients from hospitals in the south of Romania belonged to widespread international E. coli (e.g., ST131, ST10, and ST5) or K. pneumoniae (ST101) clones.
In 2020, the reported antibiotic resistance levels in Enterobacterales indicated that K. pneumoniae had the highest resistance levels in third generation cephalosporins, fluoroquinolones, aminoglycosides, and E. coli for aminopenicillins, fluoroquinolones, and third generation cephalosporins [317].
Several encoding genes and MGE (class 1 integrons and Inc plasmids) were reported in different parts of Romania (Tables 4 and 5).

Community-Acquired Antibiotic Resistance
Generally, infections are classified into two categories: community-acquired and nosocomial (intra-hospital) infections. Healthcare-associated infections are specific to admitted hospital patients and occur after at least 48 h from admission, while communityacquired infections are contracted outside of a healthcare facility and diagnosed within 48 h after admission (community onset) [346,358].
The occurrence of AR in community-acquired infections is increasing due to multiple factors. Antibiotic overuse, for example, can imbalance the composition of the gut microbiota, facilitating the emergence and colonization of the gut with antibiotic-resistant bacteria (ARB) and the proliferation of opportunistic pathogens [359]. On the other hand, antibiotic residues in the environment or in food products could select for resistance [360,361].
Community-acquired AR was scarcely investigated in Romania (Table 6). For E. coli, we have identified five studies reporting carbapenemases, ESBLs, and aminoglycosides, PMQR, trimethoprim, and tetracycline resistance genes in strains isolated from communityacquired UTIs. In A. baumannii, the presence of CHLDs, AMEs, and sulphonamide, tetracycline, and macrolide resistance genes has beeen reported, while in P. aeruginosa, the presence of MBL, AMEs, and PMQR, sulphonamide, trimethoprim, and tetracycline resistance genes has been reported in strains isolated from all geographical regions of the country. Furthermore, class 1 and 2 integrons were involved in disseminating ARGs in these strains.

Antibiotic Resistance in Veterinary Settings
Several factors can affect the occurrence and dissemination of AR in the animal industry, including antibiotic use and farm management. Many studies have focused on how the use of antibiotics in food-producing animals has led to the expansion of antibiotic resistance. In industrialized countries, the companion animal population has dramatically increased during the last few decades. The increased interaction between animals and humans leads to a higher risk of infections and the cross-transmission of AR traits. Thus, the potential of reverse zoonosis and the creation of animal reservoirs that keep the loop of infection and AR diffusion open are gaining steadily increasing concern. Antimicrobial resistance of pet origin, responsible for both direct and/or indirect threats to human health, involves mainly carbapenemase-producing enterobacteria and ESBL Gram-negative bacteria [366].
The epidemiological scale of AR transmission between humans and animals is not yet well defined, as multiple parameters should be taken into account (population features, geographical location, investigative methods), and the sole abuse or misuse of antibiotics is insufficient for such a massive transmission of resistant microorganisms between humans and pets [367,368]. Therefore, several research lines are being explored, such as humananimal transmission and vice versa, although controversial results are being observed [369]. Moreover, the environment most likely contributes to AR dissemination, intended as the vector connecting the human and animal environments, including anthropic activities. It was also suggested that monitoring non-pathogenic specimens and their potential capability to acquire resistance traits is a promising strategy to predict and prevent future resistant strains.
In Romania, very scarce information is available regarding the isolation, identification, and AR of Gram-negative bacilli from veterinary settings; the ARGs and carrying platforms were investigated only for Enterobacterales species, reported in three studies ( Table 7). The other studies reveal only the presence of resistant bacterial strains in different animal isolates without investigating the genetic background of AR.
Thus, gentamycin-and penicillin-resistant Pseudomonadaceae strains were described in samples from boar semen from three artificial insemination centers in the northwest of Romania [367]. Cristina et al. investigated the presence of AR in isolates from pet reptiles (chelonians, snakes, and lizards) and identified the presence of P. aeruginosa, Citrobacter koseri, C. brakii, and K. oxytoca resistant to cephalosporins (up to the fourth generation), tetracyclines, quinolones, aminoglycosides, and others [370]. Tîrziu et al. investigated the prevalence and AR profiles of two major foodborne pathogens (Salmonella spp. and, respectively, Campylobacter spp.) in different food products from two Transylvanian counties of Romania and revealed high levels of resistance to tetracycline, ciprofloxacin, and nalidixic acid in both pathogens [371]. A high level of AR in Campylobacter spp. was also reported in strains isolated from broiler chicken flocks from three north-western Transylvanian counties of Romania [372]. Table 7. Genetic background of AR and carrying platforms in Gram-negative strains isolated in Romanian veterinary settings. E. coli Salmonella spp.

Antibiotic Resistance in Gram-Negative ESCAPE Pathogens in Wastewaters
Antibiotics are among the most popular pharmaceuticals used in human medicine, veterinary care, and farming [376][377][378]. They are also frequent contaminants in wastewater, municipal sewage, and wastewater treatment plants' influents and effluents [379]. Hospitals generate an impressive amount of wastewater per day; the hospital effluents are loaded with pathogenic microorganisms, antibiotics, and other pharmaceutical or toxic substances, which are only partially removed during wastewater treatments, contributing to the pollution of the natural environment, including the selection and dissemination of AR [380,381]. Wastewater treatment plants (WWTPs) are one of the critical reservoirs of both antibiotic-resistant bacteria and ARGs and represent hotspots for horizontal gene transfer (HGT) via MGEs, such as plasmid integrons, transposons, resistance islands, and insertion sequences, enabling the development and dissemination of ARGs between bacteria [382,383]. Antibiotic-resistant bacteria are collected and mixed with environmental strains, which, in turn, could introduce the newly acquired ARGs into the clinics [384]. Romania had the third-highest consumption of antibacterials for systemic use in the community sector in 2019 [385]. In 2016 and 2018, the most-consumed classes for food-producing animals in Romania, according to the European Surveillance of Veterinary Antimicrobial Consumption (EVSAC), were tetracyclines and penicillins, respectively [386]. This explains the presence of antibiotics in WWTP, representing a high selective pressure for AR [387]. In this context, during the last few years, international authorities have made considerable efforts to improve the monitoring of the circulation of the antibiotic-resistant bacteria in different environments, underscoring the necessity to strengthen intersectional human, animal, and agricultural cooperation, which has been included as a priority in the work plan for the EU Health Programme. One of the priority topics of the Joint Programme Initiative on Antimicrobial Resistance (JPIAMR) is the elucidation of the role of the environment as a source for the selection and dissemination of AR, which is expected to provide essential data for monitoring AR, as the lack of surveillance is considered one of the main contributors to the spread of AR, particularly in developing countries. In this regard, one important goal is mapping the distribution of MDR strains and plasmids and different genomic lineages of critical nosocomial pathogens in different clinical and aquatic compartments. This vital knowledge could be translated into policy measures to control the emergence and spread of antibiotic-resistant bacteria [388,389].
Contrary to clinical studies, there needs to be more information regarding the ARG reservoirs in the wastewater network in Romania. Our research team showed a high repertoire of ARGs and virulence markers in K. pneumoniae ST101 isolated from intrahospital infections and wastewater samples collected from the influent and, respectively, from the effluent of hospital collecting sewage tanks in the southern regions of Romania; the transmission of MDR, carbapenemase and ESBL-producing K. pneumoniae ST101 from hospital to hospital effluent; and its persistence after the chlorine treatment [339,344]. In the country's central region, a chlorinated wastewater treatment system from a public hospital revealed the presence of the following carbapenemase and ESBL-encoding genes in the influent: bla PER , bla VIM , bla NDM-1 , and bla SHV . In contrast, the chlorinated effluent exhibited bla VIM and bla SHV [390]. Another study performed in Cluj-Napoca on one WWTP and the receiver river Somes , ul Mic revealed the presence of tetracycline and sulphonamide ARGs [sul1, tet(O), and tet(W)] in wastewater without focusing on total antibiotic-resistant bacteria or ARGs identification [391]. Butiuc-Keul et al., in 2019, revealed the genetic background of AR in Pseudomonas spp. from urban water sources and their environmental impact in north-western Romania [392]. Several carbapenemase-encoding genes have recently shown spatiotemporal variation in wastewater samples from the influent and effluent of three Cluj-Napoca WWTPs [393]. In South Romania, Van et al. recently revealed the efficiency of commercial essential oils against antibiotic-resistant P. aeruginosa clinical and wastewater strains [394]. Gheorghe-Barbu et al. have demonstrated a high repertoire of ARGs in A. baumannii and P. aeruginosa strains isolated two years consecutively from intra-hospital infections, wastewater, and surface water from three geographical regions of Romania and highlighted the importance of screening for acquired antimicrobial resistance in the environment [330] (Table 8).

Antibiotic Resistance in Other Aquatic Ecosystems
Surface water plays an essential role in AR dissemination by being both a habitat and a dissemination ecosystem for microorganisms. Recently, Banciu et al. demonstrated the dissemination of A. baumannii and P. aeruginosa clinical strains in wastewater or surface water or the presence of E. coli, K. oxytoca, C. freundii, and P. mirabilis resistant to ampicillin and clavulanic acid, strains isolated from the St. Gheorghe branch of the Danube Delta [386]. The Danube River is considered the most critical non-oceanic body of water in Europe and the "future central axis for the European Union," Its Danube Delta is included in the Biosphere Reserve and Ramsar Site lists. The Danube River crosses ten countries. This basin represents an optimal pool for resistant pathogens and anthropogenic pollutants dissemination and accumulation throughout large and distant areas, being assigned as a reservoir of AR. Previously, it has been demonstrated that Bucharest was at the top of the most polluted sampling locations from twelve WWTPs in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, and Germany) in the Danube River Basin collected and analyzed for ARGs and MGE presence [395].
Several other authors characterized at the molecular level the Gram-negative rods from the surface water (Dambovit , a river-south Romania), from the 4 Romanian natural aquatic fishery lowland salted lakes from the Natura 2000 Network located in Buzǎu and Brǎila counties, carrying a high diversity of resistance markers correlated with class one integrons [396] (see Table 8).

Conclusions
Updated information regarding the genetic background and molecular epidemiology of AR is crucial for tackling the spread of this phenomenon. This review brings together the available data regarding the AR of Gram-negative ESCAPE pathogens circulating in Romania. The big picture for Gram-negative ESCAPE pathogens reveals that all significant, clinically relevant, globally spread ARGs and carrying platforms are well established in different areas of our country and are already disseminated beyond clinical settings.
To constrain the spread of ESCAPE pathogens, it is now well recognized that collaborative efforts are required by policymakers, funders, and those responsible for the treatment and management of ESCAPE pathogens. Aside from novel drug development, these collaborative endeavors will require sustainable stewardship practices to reduce the inappropriate use of antibiotics in both the human health and agricultural sectors. In addition, improvements in factors encompassing AR surveillance, diagnostics, patient education, and patient treatment options will help facilitate AR control.