![\"MarineKAMENEFF\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2024\/12\/MarineKAMENEFF.jpg\")
<\/p>\n<\/p>\n
M. Kameneff<\/strong>, I. Marques<\/strong>, T. Lima<\/strong>, M. J. Saavedra<\/strong>, S. Anast\u00e1cio<\/strong>, G. J. Da Silva<\/strong> and E. Silveira<\/strong>*<\/p>\n <\/a><\/p>\n <\/a> ▲<\/a><\/p>\n Carbapenemase-producing (CP) strains represent a substantial global threat, deactivating carbapenems and conferring resistance to \u03b2-lactam antibiotics. They can spread across various environments, yet data on their presence in marine animals are sparse. This study aimed to assess the occurrence of carbapenemase-producing strains in wild marine animals and to analyse their antimicrobial resistance (AMR) profiles to crucial antimicrobials and heavy metals frequently encountered in the marine environment due to anthropogenic activity. A total of 28 samples were obtained from a fish auction in the Centre Region of Portugal. Non-fermenting bacilli (NFB) was isolated from the visceral content of wild marine animals. Identification of isolates was achieved through PCR-based amplification of the 16S rRNA gene, followed by sequencing. Antimicrobial susceptibility testing was conducted according to EUCAST guidelines, covering nine antimicrobials. Research of carbapenemases and metal tolerance genes was conducted by PCR, and statistical analysis utilized the Fisher\u2019s exact test. Pseudomonas<\/em> spp. and Aeromonas<\/em> spp., among other isolates were identified (n<\/em>=47\/9\/7, respectively). Susceptibility profiles showed 100% resistance or inter- mediate resistance to ticarcillin, piperacillin, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem (n<\/em>=63\/63), while 27% were resistant to meropenem (n<\/em>=17\/63) and 13% to tobramycin (n<\/em>=8\/63). All of them exhibited susceptibility to amikacin and carried multidrug resistance (MDR) profiles, including heavy metal genes (merA<\/em> and silA<\/em>). None harboured the carbapenemase genes searched (bla<\/em>KPC<\/sub>, bla<\/em>GES<\/sub>, bla<\/em>IMP<\/sub>, bla<\/em>NDM<\/sub> or\/and bla<\/em>VIM<\/sub>). In this study, MDR profiles to clinically important antimicrobials were observed, including to carbapenems. However, no carbapenemase-producing strains were identified, suggesting the presence of other genes or alternative mechanisms of resistance. These findings underscore the importance of monitoring AMR in marine ecosystems, particularly given its close ties to the food chain.<\/p>\n Key words:<\/strong> carbapenemases; multidrug-resistance; antimicrobials; heavy-metals; marine animals; Pseudomonas spp.; Aeromonas spp.<\/em><\/p><\/blockquote>\n <\/a> ▲<\/a><\/p>\n Antimicrobial resistance (AMR) has become internationally recognised as a significant global health challenge, and has the potential to disseminate across human, animal, and environmental domains, resulting in amplified human morbidity and mortality (WHO 2017; ECDC, 2022).<\/p>\n Over the last decade, the emergence and prevalence of carbapenemase-producing strains have been growing at a global scale and are directly linked to a risk factor for nosocomial infections. This pressing concern has led to their inclusion in the roster of high-priority pathogens, demanding urgent Research and Development endeavours for novel antimicrobials (WHO, 2017). Furthermore, carbapenemase-producing strains have recurrently been associated with the emergence of Multidrug-Resistant (MDR) clones, posing a substantial threat given the limited efficacy of the available antimicrobials (Nordmann and Poirel, 2019). As reported by the European Centre for Disease Prevention and Control, the highest levels of AMR in southern and eastern Europe were reported in 2020. This underscores the critical significance of early detection and identification of these strains (ECDC, 2022).<\/p>\n Carbapenemases are enzymatic proteins capable of hydrolysing carbapenem compounds along with other \u03b2-lactams, which stand out as the prevailing mechanisms behind carbapenem resistance. Notably, these enzymes have been associated with an easy spread into the community and environmental settings, mainly accomplished through mobile genetic elements, such as plasmids or transposons (Nordmann and Poirel, 2019). Such mechanisms foster horizontal gene transfer among pathogens, potentially leading to the proliferation of AMR within other bacterial ecosystems, including marine environments (Bonardi and Pitino 2019; Chen et al<\/em>., 2020; Dewi et al<\/em>., 2020; Duff et al<\/em>., 2020; Norman et al<\/em>., 2021).<\/p>\n Nonetheless, other mechanisms of resistance to carbapenems may also be documented. These include the depletion of outer membrane porins (e.g., Enterobacterales<\/em> carrying extended-spectrum beta-lactamases or AmpC enzymes), as well as the loss of OprD porins expression in Pseudomonas aeruginosa<\/em>. Furthermore, resistance might stem from the presence of efflux pumps or even due to mutations in penicillin-binding proteins (e.g., Escherichia coli<\/em>, P. aeruginosa<\/em>, and Acinetobacter baumannii<\/em>) (Botelho et al<\/em>., 2019; Nordmann and Poirel, 2019).<\/p>\n Currently, some of the most prevalent carbapenemases found in non-fermenting bacilli encompass different Ambler classes: A – Klebsiella pneumoniae<\/em> carbapenemase (KPC) and Guiana extended-spectrum-\u00df-lactamases (GES); B \u2013 Verona integron-mediated metallo-\u00df-lactamases (VIM), Active-on-imipenem (IMP) and New Delhi metallo-\u00df-lactamases (NDM), as well as class D-oxacillinases (OXA-48) (Bogaerts et al<\/em>., 2013; Nordmann and Poirel 2019). The escalation of selective pressure on bacteria finds its roots in the improper or excessive use of antimicrobials in both human and animal contexts. However, this rise in selective pressure may also be exacerbated by the presence of heavy metals within the environment (Rebelo et al<\/em>., 2021). Available data have underscored the potential role of metal tolerance in contributing to the emergence of MDR bacteria, primarily through an indirect selection process. Heavy metals naturally exhibit a widespread presence in marine ecosystems. However, it is primarily anthropogenic activities that serve as the principal contributors, emanating from sources like metal-laden food additives, agricultural practices related to biosecurity and hygiene, industrial and mining operations, as well as wastewater discharges (Tappin et al<\/em>., 2010; Romero et al<\/em>., 2017; Rebelo et al<\/em>., 2021). Scientific data has shown that wild marine animals, even in the absence of direct antimicrobial treatment, could serve as potential reservoirs for resistant bacterial strains capable of infecting or colonising other animals or humans (Wallace et al<\/em>., 2013; Marti et al<\/em>., 2018; Duff et al<\/em>., 2020; Norman et al<\/em>., 2021). Undoubtedly, wild marine animals possess the capacity to accumulate heavy metals, a factor that could potentially facilitate the acquisition of AMR genes and lead to the emergence of a reservoir of MDR bacteria, including carbapenemase-producing strains. This scenario poses a significant threat to the safety and quality of marine water, consequently endangering public health (Woodford et al<\/em>., 2018; Botelho et al<\/em>., 2019; Figueiredo et al<\/em>., 2019). The point is emphasised that AMR has been scarcely documented in wild marine animals, highlighting their crucial role as sentinels of marine well-being and health (Norman et al<\/em>., 2021).<\/p>\n This study seeks to provide novel perspectives on the prevalence of AMR, with emphasis on carbapenemase-producing organisms in wild marine animals along the central Portuguese coast, to assess the impact of anthropogenic activities on their potential dispersion within the marine ecosystem.<\/p>\n <\/a> ▲<\/a><\/p>\n Between May and April 2022, a total of 28 samples were collected from visceral tissues of wild marine fishes: Trachurus trachurus<\/em> (n<\/em>=2), Dicentrarchus labrax<\/em> (n<\/em>=1), Trisopterus luscus<\/em> (n<\/em>=1), Zeus faber<\/em> (n<\/em>=8), Diplodus <\/em>sargus (n<\/em>=6), Merluccius merluccius<\/em> (n<\/em>=4), Todarodes sagittatus<\/em> (n<\/em>=3), Scyliorhinus canicula<\/em> (n<\/em>=2) and Raja brachyura<\/em> (n<\/em>=1). All fish were captured within the Northern Atlantic waters of Portugal\u2019s Central Region. The specimens were conveyed within a regulated refrigerated setting and expeditiously subjected to processing upon their reception at the microbiology laboratory. For the selection of the carbapenemase-producing strains, the samples were plated onto ChromID\u00ae<\/sup> Carba Smart Agar (Biomerieux), and incubated for 18-24 hours at 35\u00b12\u00b0C. The selection of representative colonies was chosen according to Woodford et al<\/em>. (2018). Distinctive colonies were selected and cultivated on MacConkey Agar (Oxoid), followed by isolation on Trypticase Soy Agar (Liofilchem) to achieve a state of pure culture.<\/p>\n The identification of representative and suspected carbapenemase-producing strains was accomplished using the amplification and sequencing of the 16S rRNA gene as previously described (H\u00e9ritier et al<\/em>., 2003). Sequencing was conducted by Eurofins (https:\/\/www.eurofins.pt\/), and the analysis was carried out using the NCBI Reference Sequence Database and the Basic Local Alignment Search Tool (https:\/\/blast.ncbi.nlm.nih.gov).<\/p>\n Antimicrobial susceptibility testing was conducted using the standard disk diffusion method on Mueller Hinton agar (Biolab\u00ae<\/sup>), following the guidelines outlined by the European Committee on Antimicrobial Susceptibility Testing (European Committee on Antimicrobial Susceptibility Testing, 2022). This process involved the assessment of nine antimicrobials: piperacillin (PRL, 30 \u03bcg), piperacillin-tazobactam (TZP, 30-6 \u03bcg), ticarcillin (TIC, 75 \u03bcg), ceftazidime (CAZ, 10 \u03bcg), imipenem (IMP, 10 \u03bcg), meropenem (MEM, 10 \u03bcg), ciprofloxacin (CIP, 5 \u03bcg), amikacin (AK, 30 \u03bcg) and tobramycin (TOB, 10 \u03bcg) (Oxoid\u00ae<\/sup>). MDR profiles were deemed when bacteria exhibited resistance to at least three or more antimicrobials from different categories (Magiorakos et al<\/em>., 2012).<\/p>\n Isolates resistant to carbapenems and suspected to be carriers of the most prevalent carbapenemases were selected for PCR screening for bla<\/em>GES<\/sub>, bla<\/em>VIM<\/sub>, bla<\/em>IMP<\/sub>, bla<\/em>NDM<\/sub>, bla<\/em>KPC<\/sub> genes as previously described (Table 1) (Bogaerts et al<\/em>., 2013).<\/p>\n Genes linked to bacterial tolerance of heavy metals were chosen according to their potential occurrence in marine environment and subsequently probed through PCR: pcoA<\/em> (periplasmic multicopper oxidase), pcoD<\/em> (copper inner membrane pump); silA<\/em> (efflux pump), silE<\/em> (periplasmic silver-sequestration protein); arsB<\/em> (efflux pump); and merA<\/em> (mercuric reductase) (Table 1) (Mour\u00e3o et al<\/em>., 2015).<\/p>\n <\/a> ▲<\/a><\/p>\n Colonies growing on ChromID\u00ae<\/sup> Carba Smart Agar displayed morphological characteristics that did not align with the descriptions provided in the manufacturer\u2019s instructions. Consequently, the selected isolates predominantly appeared colourless (n<\/em>=47), while a smaller number exhibited shades of brown (n<\/em>=9), red (n<\/em>=2), and green\/greenish (n<\/em>=5) colouration.<\/p>\n Of all the representative isolates (n<\/em>=56), identification was performed through sequencing of the 16S rRNA gene. These isolates were categorised into Pseudomonas<\/em> spp. (n<\/em>=47, colourless colonies) and Aeromonas<\/em> spp. (n<\/em>=9, brownish colonies). The remaining colonies, exhibiting varying shades of red and greenish tones, were categorised as non-fermenting bacilli (NFB) (n<\/em>=7) and were excluded from sequencing.<\/p>\n The antimicrobial susceptibility testing results revealed resistance or intermediate resistance among the isolates to all \u03b2-lactams, including subclasses such as penicillins, cephalosporins, and carbapenems, as well as to fluoroquinolones. Among the 63 isolates obtained from the visceral samples of wild marine fish, 100% (n<\/em>=63\/63) exhibited resistance or intermediate resistance to TIC, PRL, TZP, CAZ, IMP, and CIP, followed by MEM (27%, n<\/em>=17\/63: Pseudomonas<\/em> spp., n<\/em>=16; Aeromonas<\/em> spp., n<\/em>=1), and TOB (13%, n<\/em>=8\/63: Aeromonas<\/em> spp., n<\/em>=3; other NFB, n<\/em>=3 and Pseudomonas<\/em> spp., n<\/em>=2). Notably, all isolates demonstrated susceptibility to AK (Figure 1).<\/p>\n The findings unveiled seven distinct MDR profiles (heavy metal tolerance genes were included) (Table 2). <\/p>\n Noteworthy, 61.9% of the isolates shared the MDR profile \u201cTIC, PRL, TZP, CAZ, IMP and CIP\u201d (n<\/em>=32\/63: Pseudomonas<\/em> spp., Aeromonas<\/em> spp., and NFB; n<\/em>=22\/6\/4, respectively), followed by \u201cTIC, PRL, TZP, CAZ, IMP, MEM and CIP\u201d (n<\/em>=10\/63: Pseudomonas<\/em> spp., n<\/em>=10), \u201cTIC, PRL, TZP, CAZ, IMP, TOB and CIP\u201d (n<\/em>=7\/63: Pseudomonas<\/em> spp., Aeromonas<\/em> spp., and NFB, n<\/em>=2\/2\/3, respectively). Lastly, the final profile was identified in just one isolate of Aeromonas<\/em> spp. and exhibited resistance to all tested antimicrobial families, except for amikacin (penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones) (Figure 2).<\/p>\n Additionally, among the entire isolate pool, 9.5% (n<\/em>=6\/63) and 6.4% (n<\/em>=4\/63) of Pseudomonas<\/em> spp. were found to harbour the merA<\/em> gene within two distinct MDR profiles: i) \u2018TIC, PRL, TZP, CAZ, IMP, and merA<\/em> or ii) \u2018TIC, PRL, TZP, CAZ, IMP, MEM, CIP, and merA<\/em>. Additionally, the gene silA<\/em> was identified in a sole profile, constituting 4.8% (n<\/em>=3\/63) of the isolates, characterised as TIC, PRL, TZP, CAZ, IMP, CIP, and silA<\/em>\u201d (Table 2, Figure 2).<\/p>\n After conducting a PCR-based screening to detect the presence of carbapenemase genes (bla<\/em>KPC<\/sub>, bla<\/em>GES<\/sub>, bla<\/em>IMP<\/sub>, bla<\/em>NDM<\/sub> or bla<\/em>VIM<\/sub>) in isolates exhibiting resistance to carbapenems (Pseudomonas<\/em> spp., Aeromonas<\/em> spp., and others NFB: n<\/em>=3\/2\/1, respectively), it became evident that none of the isolates harboured these carbapenem resistant genes.<\/p>\n In the search for metal tolerance genes, it was noted that 15.9% (n<\/em>=10\/63) of Pseudomonas<\/em> spp. carried the merA<\/em> gene, while 4.8% (n<\/em>=3\/63) exhibited the silA<\/em> gene. The presence of silE<\/em>, pcoA<\/em>, pcoD<\/em>, and arsB<\/em> genes, responsible for conferring tolerance to silver, copper, and arsenic, was not observed.<\/p>\n <\/a> ▲<\/a><\/p>\n The culture media ChromID\u00ae<\/sup> Carba Smart Agar is made to select Enterobacterales<\/em> carbapenemase-producing from human clinical specimens. Using samples from the marine environments, as in this study, the conditions of growth may vary, since the bacterial strains may have other individual requirements. Nevertheless, it allowed for the recovery of a high rate of carbapenem-resistant or intermediated-resistant isolates. Following the manufacturer\u2019s instructions, the emergence of other MDR bacteria is possible, which could explain the growth of Pseudomonas<\/em> spp., Aeromonas<\/em> spp., and other NFB instead of Enterobacterales<\/em>. Indeed, the significant occurrence of Pseudomonas<\/em> spp. observed in this study may be linked to their inherent presence within the microbiota of carnivorous, omnivorous, and planktivorous fish. This prevalence is further reinforced by their widespread distribution across aquatic environments (Egerton et al<\/em>., 2018).<\/p>\n Despite scientific literature documenting that wild marine life can be an important reservoir of AMR genes and could play a significant role in their worldwide dissemination, especially through migratory animals, this is the first study to report high levels of resistance or intermediate resistance to critical or highly important antimicrobials in wild marine fish (Marti et al<\/em>., 2018; Chen et al<\/em>., 2020; Fernandes et al<\/em>., 2021; Norman
\nMarine KAMENEFF<\/strong>, DVM, MSc, Department of Veterinary Sciences (DVS) – University School Vasco da Gama (EUVG), 3020-210 Coimbra, Portugal; In\u00eas MARQUES<\/strong> DVM, MSc, DVS-EUVG, 3020-210 Coimbra, Portugal; Tiago LIMA<\/strong>, Pharm MSc, PhD, DVS-EUVG 3020-210 Coimbra, Portugal, Faculty of Pharmacy, University of Coimbra (FFUC), Centre for Innovative Biomedicine and Biotechnology, University of Coimbra (CIBB-UC), 3000-548 Coimbra, Portugal; Maria Jos\u00e9 SAAVEDRA<\/strong>, Animal Science PhD, Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB-Inov4Agro), Animal and Veterinary Research Center (CECAV-AL4AnimalS), University of Tr\u00e1s-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal; Sofia ANAST\u00c1CIO<\/strong>, DVM, MSc, Pharm PhD, DVS-EUVG 3020-210 Coimbra, Portugal, CIBB-UC, 3000-548 Coimbra, Portugal; Research Center Vasco da Gama (CIVG), 3020-210 Coimbra, Portugal, University Institute of Health Sciences, CESPU, CRL, 4585-116 Gandra, Portugal; Gabriela Jorge DA SILVA<\/strong>, PharmD, PhD, FFUC, CIBB-UC, 3000-548 Coimbra, Portugal; Eduarda SILVEIRA<\/strong>* (Corresponding author, e-mail: eduardamorenosilveira@gmail.com\/eduardasilveira@euvg.pt), PharmD, PhD, DVS-EUVG 3020-210 Coimbra, Portugal, FFUC, CIBB-UC, 3000-548 Coimbra, Portugal, CIVG, 3020-210 Coimbra, Portugal, Research Center for Natural Resources, Environment and Society (CERNAS), 3045-601 Coimbra, Portugal<\/div>\n![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/03\/pdf.png\")
<\/a>https:\/\/doi.org\/10.46419\/vs.56.3.4<\/a><\/div>\n<\/p>\n\n
\nAbstract<\/a>Introduction<\/a>Methods<\/a>Results<\/a>Discussion<\/a>Ethical approval<\/a>Acknowledgments<\/a>Financial support and sponsorship<\/a>References<\/a>Sa\u017eetak<\/a><\/span><\/div>\n<\/div>\n\n
Abstract<\/h2>\n
\nIntroduction<\/h2>\n
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\nThis phenomenon arises due to the co-location of genes responsible for both metal and antibiotics tolerance\/resistance within the same mobile genetic elements, such as plasmids (Romero et al<\/em>., 2017; Figueiredo et al<\/em>., 2019).<\/p>\nMethods<\/h2>\n
\nStudy design<\/h3>\n
\nThis procedural sequence entailed the extraction of visceral contents, followed by buffered peptone water pre-enrichment. Subsequently, the samples were subjected to a homogenisation process (Interscience\u00ae<\/sup> BagMixer\u00ae<\/sup>), followed by overnight incubation for 16-24 hours at 35\u00b12\u00b0C.<\/p>\nBacterial selection and growing conditions<\/h3>\n
Identification of isolates<\/h3>\n
Study of antimicrobial susceptibility<\/h3>\n
Study of carbapenemases and heavy metal susceptibility<\/h3>\n
![\"Table01-Assessing\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2024\/12\/Table01-Assessing.png\")
Legend<\/strong>: FW \u2013 Forward; RV \u2013 Reverse.<\/figcaption><\/figure>\nResults<\/h2>\n
\nBacterial selection<\/h3>\n
Identification of isolates<\/h3>\n
Study of antimicrobial susceptibility<\/h3>\n
![\"Figure01-Assessing\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2024\/12\/Figure01-Assessing.webp\")
Legend<\/strong>: P<\/em><0.05 (Fisher\u2019s exact test) Ticarcillin (TIC), piperacillin (PRL), piperacillin-tazobactam (TZP), ceftazidime (CAZ), imipenem (IMP), tobramycin (TOB), amikacin (AK), meropenem (MEM), ciprofloxacin (CIP).<\/figcaption><\/figure>\n![\"Table02-Assessing\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2024\/12\/Table02-Assessing.png\")
Legend<\/strong>: HMTG \u2013 Heavy Metal Tolerance genes; Ticarcillin (TIC), piperacillin (PRL), piperacillin-tazobactam (TZP), ceftazidime (CAZ), imipenem (IMP), tobramycin (TOB), meropenem (MEM), ciprofloxacin (CIP); isolates showing intermediate susceptibility were categorised as resistant.<\/figcaption><\/figure>\n![\"Figure02-Assessing\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2024\/12\/Figure02-Assessing.webp\")
Legend<\/strong>: Profile I<\/strong>: TIC, PRL, TZP, CAZ, IMP, CIP (n<\/em>=32\/63); Profile II<\/strong>: TIC, PRL, TZP, CAZ, IMP, CIP, merA (n<\/em>=4\/63); Profile III<\/strong>: TIC, PRL, TZP, CAZ, IMP, CIP, silA (n<\/em>=3\/63); Profile IV<\/strong>: TIC, PRL, TZP, CAZ, IMP, MEM, CIP (n<\/em>=7\/63); Profile V<\/strong>: TIC, PRL, TZP, CAZ, IMP, TOB, CIP (n<\/em>=10\/63); Profile VI<\/strong>: TIC, PRL, TZP, CAZ, IMP, MEM, CIP, merA (n<\/em>=6\/63); Profile VII<\/strong>: TIC, PRL, TZP, CAZ, IMP, TOB, MEM, CIP (n<\/em>=1\/63).
Isolates showing intermediate susceptibility were categorised as resistant.<\/figcaption><\/figure>\nStudy of carbapenemase and heavy metal susceptibility<\/h3>\n
Discussion<\/h2>\n
\n