https://orcid.org/0000-0001-7332-1647
Figures
Abstract
The impact of antimicrobial resistance (AMR) on global public health has been widely documented. AMR in the environment poses a serious threat to both human and animal health but is frequently overlooked. This study aimed to characterize the association between phenotype and genotype of AMR, virulence genes and Extended-Spectrum β-Lactamase (ESBL) production from estuarine environment. The Salmonella (n = 126) and E. coli (n = 409) were isolated from oysters and estuarine water in Thailand. The isolates of Salmonella (96.9%) and E. coli (91.4%) showed resistance to at least one antimicrobial agent. Multidrug resistance (MDR) was 40.1% of Salmonella and 23.0% of E. coli. Resistance to sulfamethoxazole was most common in Salmonella (95.2%) and E. coli (77.8%). The common resistance genes found in Salmonella were sul3 (14.3%), followed by blaTEM (11.9%), and cmlA (11.9%), while most E. coli were blaTEM (31.5%) and tetA (25.4%). The ESBL production was detected in Salmonella (1.6%, n = 2) of which one isolate was positive to blaTEM-1. Eight E. coli isolates (2.0%) were ESBL producers, of which three isolates carried blaCTX-M-55 and one isolate was blaTEM-1. Predominant virulence genes identified in Salmonella were invA (77.0%), stn (77.0%), and fimA (69.0%), while those in E. coli isolates were stx1 (17.8%), lt (11.7%), and stx2 (1.2%). Logistic regression models showed the statistical association between resistance phenotype, virulence genes and ESBL production (p < 0.05). The findings highlighted that estuarine environment were potential hotspots of resistance. One Health should be implemented to prevent AMR bacteria spreading.
Citation: Jeamsripong S, Kuldee M, Thaotumpitak V, Chuanchuen R (2023) Antimicrobial resistance, Extended-Spectrum β-Lactamase production and virulence genes in Salmonella enterica and Escherichia coli isolates from estuarine environment. PLoS ONE 18(4): e0283359. https://doi.org/10.1371/journal.pone.0283359
Editor: Iddya Karunasagar, Nitte University, INDIA
Received: October 20, 2022; Accepted: March 7, 2023; Published: April 28, 2023
Copyright: © 2023 Jeamsripong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw data was available at https://figshare.com/articles/dataset/Antimicrobial_Resistance_Extended-Spectrum_-Lactamase_Production_and_Virulence_Genes_in_Salmonella_enterica_and_Escherichia_coli_Isolates_from_Estuarine_Environment/21761042
Funding: This study was supported by the Thailand Science Research and Innovation (TSRI) (CU_FRB640001_01_31_9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Antimicrobial resistance (AMR) has been recognized as one of the greatest challenges endangering the health of people, animals, and the environment. One Health approach has been applied for managing and controlling AMR at national and international levels. The Unites States Center for Disease Control and Prevention (U.S. CDC) estimated that greater than 2.8 million people and at least 35,000 deaths are affected by AMR in the U.S. annually [1]. Without a global response to AMR, it has been predicted that AMR could cause 10 million deaths annually by 2050 [2]. The global action plan to tackle AMR lists strengthening knowledge through AMR surveillance as one of the important measures to address the global AMR issue [3]. However, AMR monitoring and surveillance in the environment is rather limited and not harmonized due to several pathways responsible for AMR releasing to the environment. Therefore, knowing the magnitude of the AMR in the environment is needed for estimating impact on human and animal health.
Salmonella spp. is one of the most frequently isolated foodborne pathogens and a major public health threat worldwide. Humans usually get infected with Salmonella by consumption of contaminated food and water. Different food commodities, including poultry, swine, fish, shellfish, and produce were linked to salmonellosis in humans [4]. Salmonellosis causes 93.8 human cases and almost 155,000 deaths annually [5]. Salmonella contains many virulence factors that play a crucial role in the ability to infect the host cells and propagate. Salmonella virulence factors enhancing pathogenesis include Salmonella pathogenicity islands (SPIs), Salmonella virulence plasmids, pili, and enterotoxin [6, 7]. Despite a particular public health concern, knowledge of abundance of genotypic diversity of resistant and virulent Salmonella isolated from the environment is still limited.
Bivalve mollusks serve as useful and practical bioindicators of environmental fecal contamination. They have the capacity to accumulate nutrients, chemicals, and various microorganisms [8]. E. coli has been used to monitor fecal contamination and AMR in bacteria from food-producing animals for public purposes [9, 10]. Resistant E. coli can spread and transfer their resistance determinants to inter- and intra-bacterial species. A previous metagenomic analysis in untreated sewage revealed that multidrug resistance (MDR) bacteria were commonly found in waste disposed to the environment [11]. Therefore, the estuarine environment is the area of particular concern due to the high diversity and abundance of resistant bacteria that potentially pose a significant public health threat [12].
Extended-Spectrum β-Lactamases (ESBLs) are a group of enzymes that confer resistance to penicillins, third and fourth generation cephalosporins, and monobactams [13]. The spread of various types of ESBL-producing E. coli and Salmonella has been reported in different sectors, including human, livestock animals, and aquaculture. The genes encoding of ESBL are associated with mobile genetic elements, which can horizontally transfer to other bacterial species [14]. Infection of ESBL-producing bacterial pathogens in patients has been increasingly reported to be associated with treatment failure and increase morbidity and mortality rates due to limited effective antibiotics. Therefore, the objectives of this study were to examine phenotypic and genotypic AMR, virulence genes, and ESBL production, and to build statistical models of the association between the most common resistance phenotype and other resistance phenotype and genotypes, virulence genes, and ESBL production among Salmonella and E. coli isolated from oyster and estuarine water samples.
Materials and methods
Bacterial strains
Salmonella (n = 126) and E. coli (n = 409) isolates were collected from stored collection strains in the Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University. All isolates were stored in 20% glycerol stock solution at -80°C. The Salmonella isolates were collected from oysters (n = 123) and estuarine water (n = 3), whereas the E. coli isolates were retrieved from oysters (n = 250) and estuarine water (n = 159) samples. Oysters and estuarine waters were collected monthly between April 2016 and March 2017 from Phang Nga province in southern Thailand as previously described [15]. The oysters were wild caught, and not exposed to antimicrobials.
Antimicrobial susceptibility testing
The agar dilution method was performed to determine minimum inhibitory concentrations (MICs) according to the Clinical and Laboratory Standard Institute [16]. Eight antimicrobials and their breakpoints were ampicillin (32 µg/ml), chloramphenicol (32 µg/ml), ciprofloxacin (4 µg/ml), gentamicin (8 µg/ml), streptomycin (32 µg/ml), sulfamethoxazole (512 µg/ml), tetracycline (16 µg/ml) and trimethoprim (16 µg/ml). E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 were used for quality control. The multidrug resistance (MDR) was classified as resistance to at least three groups of antimicrobials.
Detection of AMR gene
All isolates were tested for the presence of AMR genes including genes represented to ampicillin (blaTEM), chloramphenicol (catA, catB and cmlA), quinolone (qnrA, qnrB, and qnrS), aminoglycosides (acc(3)IV and aadA1), streptomycin (strA and strB), tetracycline (tetA and tetB), sulfamethoxazole (sul1, sul2, and sul3), and trimethoprim (dfrA1 and dfrA12) (Table 1). Conventional PCR was performed to detect most AMR genes, except genes corresponding to quinolone and sulfamethoxazole, which were used multiplex PCR. DNA templates of all E. coli and Salmonella were prepared using whole cell boiling technique [17]. Toptaq PCR Master Mix Kit (Merck, Munich, Germany) were followed as manufacturer’s instruction. The PCR products were separated by gel electrophoresis using 1.5% agarose gel in 1X Tris-acetate/EDTA. Gels were stained with Redsafe™ Nucleic Acid Staining Solution (iNtRon Biotechnology, Seongnam, South Korea) and visualized PCR products under UV light using Omega Fluor™ gel documentation system.
Phenotypic and genotypic detection of ESBL production
Disk diffusion method was used to examine ESBL production followed by CLSI standard [16]. The detection of ESBL production consists of screening and confirmation tests. Ceftazidime (30 µg), cefotaxime (30 µg), and cefpodoxime (10 µg) were used for initial screening. All isolates that showed resistance to at least one of cephalosporins were further confirmed using a combination disk diffusion method using cephalosporins combination with clavulanic acid. The positive ESBL production was interpreted by determining the difference of inhibition zone between solely cephalosporin and cephalosporin combine with clavulanic acid. The positive ESBL-production isolates were identified β-lactamases genes (blaTEM, blaSHV, blaCMY-2, and blaCTX-M-55). The blaTEM gene was examined using conventional PCR, while blaSHV, blaCMY-2, and blaCTX-M-55 were using multiplex PCR with the specific primers as described in Table 2.
Nucleotide sequence
PCR amplicons of positive ESBL production isolates were purified using GeneJET PCR purification kit (Thermo Fisher Scientific, Vilnius, Lithuania) and submitted for DNA sequencing (Bionics Co., ltd., Gyeonggi-Do, Republic of Korea). The result of the DNA sequence was blasted and aligned with references embedded in GenBank database available from the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST) (accession number OQ282894-OQ282896).
Detection of virulence genes
Virulence genes of Salmonella, including invasin (invA), fimbrial protein (fimA), and enterotoxin (stn) genes were observed (Table 3). Heat-labile toxin (lt), heat-stable toxin (st), STEC (stx1 and stx2) and EPEC for attaching and effacing protein (eae) were examined in all E. coli isolates. Most of virulence genes were detected using conventional PCR. The detection of stx1 and stx2 genes was performed by multiplex PCR.
Statistical analyses
Descriptive statistics were performed to identify prevalence of resistance phenotype and genotype, resistance pattern, MDR, virulence genes, and ESBL production of E. coli and Salmonella isolates. Logistic regression analysis was used to examine the association among AMR, virulence genes and ESBL production. The dependent variable was the highest resistance rate, and independent variables included resistance genes, resistance phenotype, virulence genes, ESBL production and MDR. A p-value and confidence intervals of regression analyses were adjusted for potential correlated data within type of sample (oysters and estuarine waters) using robust variant estimator. Univariate analysis was performed to screen for potential significance of predictors. Forward selection and backward elimination were used to select potential candidates for multivariable analysis. Final regression models of E. coli and Salmonella were received based on p < 0.05 and likelihood ratio test. All statistical analyses were performed using Stata 14.0 (StataCorp, TX, USA). Two-sided hypothesis tests were used with 5% of significant level.
Results
Phenotype of AMR in Salmonella and E. coli isolates
The resistance rate of Salmonella (n = 126) and E. coli (n = 409) isolates were presented (Table 4). Salmonella resistant to at least one antibiotic was reported almost 70% (n = 125/129). The prevalence of MDR Salmonella was 23.0% (n = 29). The most prevalence of AMR in Salmonella isolates was sulfamethoxazole (95.2%, n = 120/126), followed by trimethoprim (37.3%, n = 47/126), and ampicillin (36.5%, n = 46/126). The AMR pattern found in Salmonella isolates were SUL (37.3%, n = 47/126), AMP-SUL-TET-TRI (11.1%, n = 14/126), and SUL-TRI (10.3%, n = 13/126), respectively.
Of all E. coli isolates, 94.1% (n = 385/409) were resistant to at least one antibiotic, while the prevalence of MDR was observed at 40.1% (n = 164/409). Only 24 E. coli isolates (5.9%) were susceptible to all tested antibiotics. The predominant AMR prevalence were sulfamethoxazole (77.8%, n = 318/409), ampicillin (55.3%, n = 226/409), and tetracycline (40.1%, n = 164/409), respectively. The most common resistance patterns of E. coli (n = 409) were SUL (24.0%, n = 98/409), followed by AMP-CHO-STR-SUL-TET-TRI (6.8%, n = 28/409), AMP (5.9%, n = 24/409), and AMP-SUL (5.4%, n = 22/409).
The presence of AMR genes in Salmonella and E. coli isolates
The Salmonella isolates (n = 126) from oyster and estuarine water were harbored sul3 (14.3%, n = 18), followed by blaTEM (11.9%, n = 15), cmlA (11.9%, n = 15), tetA (11.1%, n = 14), and dfrA12 (9.5%, n = 12), while catA, catB, qnrA, aac(3)IV, aadA1, strB, tetB, sul1, sul2, and dfrA1 were not observed (Table 5). The blaTEM-1 (31.5%, n = 129), tetA (25.4%, n = 104), and strA (14.9%, n = 61) were predominant resistance genes in the E. coli isolates, while qnrA, aac(3)IV, and strB were absent (Table 5).
ESBL production in Salmonella and E. coli isolate
For all Salmonella isolates, two (1.6%) isolates from oyster samples (serovars Augustenborg and II) (data not shown) were confirmed as ESBL producing isolates. The serovar II of ESBL-producing Salmonella was blaTEM-1 positive. Eight (2.0%) out of 409 E. coli isolates from estuarine water were ESBL-producers, of which three isolates were positive for blaCTX-M-55. None of ESBL-producing E. coli isolates was retrieved from oyster samples.
Distribution of virulence genes
Out of 409 E. coli isolates, 50.0% of lt, 41.1% of stx1 and 40% of stx2 were MDR. Of all Salmonella isolates, invA (77.0%, n = 97/126), stn (77.0%, n = 97/126), and fimA (69.0%, n = 87/126) were the frequently found virulent genes (Table 6). Among these, the Salmonella isolates positive to invA (28.9%), fimA (29.9%), and stn (26.8%) were MDR. The E. coli isolates mainly harbored stx1 (17.8%, n = 73/409), followed by lt (11.7%, n = 48/409) and stx2 (1.2%, n = 5/409) (Table 6). The E. coli isolates from oyster samples predominantly contained stx1 (10.3%) and lt (9.5%), while the isolates from estuarine water commonly carried stx1 (7.6%). The st and stx2 genes were not detected in E. coli isolated from estuarine water.
Co-existence among AMR, ESBL production, and virulence genes
One Salmonella isolated from oyster sample harbored blaTEM-1 with MDR to ampicillin, chloramphenicol, sulfamethoxazole, trimethoprim, and tetracycline. The latter was also positive to invA, sul3, cmlA, and dfrA12 genes. An ESBL-producing E. coli isolated from cultivation water harbored blaTEM-1 was resistant to ampicillin, while three ESBL-producing E. coli with blaCTX-M-55 were MDR.
Association between AMR, virulence genes, ESBL production, and MDR
Based on logistic regression analyses, the sulfamethoxazole-resistant Salmonella were statistically associated with the presence of ampicillin resistance (OR = 3.06), trimethoprim resistance (OR = 1.47), and invA (OR = 1.95) (p < 0.0001) compared with those isolates that were not resistant to sulfamethoxazole. The sulfamethoxazole-resistant Salmonella were negatively associated with ESBL production (p < 0.0001, OR = 0.02) and stn (p < 0.0001, OR = 0.56).
The E. coli isolates resistant to sulfamethoxazole were positively associated with the presence of trimethoprim (p = 0.027, OR = 1.55), ESBL production (p < 0.0001, OR = 1.83), MDR (p = 0.008, OR = 10.33), addA1 (p = 0.002, OR = 3.05), strA (p = 0.044, OR = 2.66), and sul3 (p < 0.0001, OR = 8.38) than other isolates that were susceptible to sulfamethoxazole (Table 7). However, the E. coli isolates resistant to sulfamethoxazole were negatively associated with two virulence genes, including lt (p = 0.025, OR = 0.43) and stx (p = 0.022, OR = 0.41), and dfrA12 (p < 0.0001, OR = 0.10).
Discussion
One of the main findings of this study is more than 90% of Salmonella and E. coli from fresh oyster (96.7%; n = 119/123 of Salmonella and 95.2%; n = 238/250 of E. coli isolates) and estuarine water (100.0%; n = 3/3 of Salmonella and 92.5%; n = 147/159 of E. coli isolates) samples were resistant to at least one antimicrobial. MDR Salmonella (23.0%) and E. coli (40.0%) were also isolated, even though the oysters were received from wild caught with no evidence of antimicrobial use. This cultivation site could be contaminated from nearby communities and agriculture according to previous studies [29, 30], so that trackback investigation to identify the source of AMR in estuarine environment is recommended. Estuarine water was considered a potential hotspot to surveillance of AMR distribution in the environment [30]. Humans can be infected with AMR bacteria by eating aquatic animals or direct contact with contaminated environment. Resistant bacteria found in this study are considered as important estuarine environmental pollutants that can adversely affect food security and public health.
In this study, the highest resistance rates acquired by both Salmonella (95.2%) and E. coli (77.8%) was to sulfamethoxazole. However, a previous study reported the lower prevalence of Salmonella resistant to sulfonamides (56.5%) in retail aquaculture products such as shellfish, calm, fish, shrimp, and others in Shanghai [31]. The high prevalence of sulfamethoxazole observed in this study may be widely used in human and animal medicine because this antimicrobial can be used to treat and prevent many bacterial infections at affordable cost [32].Therefore, it is possible that sulfamethoxazole could disseminate and accumulate to the environment. Sulfamethoxazole is effective against both Gram-negative and Gram-positive bacteria, including E. coli and Listeria monocytogenes. This antimicrobial agent is commonly used to treat urinary tract infection, bronchitis, and prostatitis. In veterinary medicine, sulfonamides have been used in swine and cattle production for treatment of urinary and respiratory tract infection. High concentration of sulfonamides in the environment has been indicated in livestock manure due to the common use of this antimicrobial [33, 34]. Sulfamethoxazole-resistant bacteria was also found in surface water and soil causing environmental pollutants as a result of the widely used in treatment of animals and humans [35]. The impact of sulfonamides contamination in the environment could result in hazardous to human health (e.g., difficult to treat of resistant bacteria, prolong hospital stay etc.), and alter microbial community [33]. However, the consequences of sulfonamide contamination in the ecosystem were still unclear [36]. Therefore, the removal of these resistant bacteria from healthcare facilities, livestock farms, and communities are needed to reduce the contamination to the coastal environment. Previous studies developed the removal of sulfonamides by using anaerobic membrane bioreactor in swine wastewater, and the use of Pleurotus eryngii for degradation of sulfonamides [37, 38].
Besides sulfamethoxazole resistance, the high resistance was observed for trimethoprim (37.3%) and ampicillin (36.5%) in Salmonella, and for ampicillin (55.3%), and tetracycline (40.1%) in E. coli. These findings agreed with previous studies conducted in aquatic animals and estuarine environment [39, 40]. High resistance rates to ampicillin (100%) and erythromycin (83.33%) in Salmonella isolates were previously reported in water and sediment [41]. The high resistance to sulfamethoxazole, ampicillin, tetracycline, and trimethoprim observed in this study was commonly reported in humans and animals [42–45]. In Thailand, the molecular epidemiology and association of AMR among of E. coli and S. enterica have been extensively investigated from pigs, pork, and humans indicating the potential risk of AMR spreading [43, 46]. Even though the precise genetic relationship information is still lacking, the observations of resistance to these antimicrobials in humans, food-producing animals, and environment in the same country confirm that AMR is a complex One Health issue.
S. enterica serovar Paratyphi B causes a serious disease, Paratyphoid, in humans. The serovars Paratyphi B poses a significant health risk due to being associated with sporadic outbreaks of human infection and multistage outbreaks of seafood products [46–48]. The symptoms of paratyphoid infection in humans are fever, loss of appetite, weakness, headaches, diarrhea, and may be a life-threatening multi-systemic illness. The pathogens were recently isolated from poultry and poultry meat from Europe and Latin America [49]. A study reported that serovar Paratyphi B was isolated from oysters (22.7%) in Thailand [15]. In this study, the serovars Paratyphi B was isolated from oysters (13.5%, n = 17/126), and all these isolates were resistant to at least one antimicrobial and 29.4% (n = 5/17) were MDR. More than 75% (n = 13/17) of these isolates contained virulence genes (i.e., fimA and stn), and 64.7% (n = 11/17) of all Paratyphi B isolates harbored invA. The presence of MDR Paratyphi B isolates in oysters may pose a serious threat to public health in the near future due to the difficulty in controlling strategic action.
Most resistance genes detected in this study corresponded well to observed resistance phenotype, suggesting that resistance genes were usually expressed when present. In Salmonella isolates, the most detected resistance genes were sul3 (14.3%), blaTEM (11.9%), cmlA (11.9%), and tetA (11.1%), while those in E. coli isolates were blaTEM (31.5%), followed by tetA (25.4%) and strA (14.9%). High prevalence (91.3%) of blaTEM gene was previously reported in oysters [50], which agreed with this study. This study observed the presence of β-lactamase encoding blaTEM-1 indicating a narrow spectrum activity against β-lactamase of E. coli and Salmonella. This indicated that the estuarine environment serves as a potential hotspot of AMR bacteria carrying resistance determinants that may be transferred to bacterial pathogens in humans and animals.
In this study, the occurrence of ESBL-producing E. coli (2.0%) and Salmonella (1.6%) was lower than in a previous study, which greatly varied in humans (11–72%), animals (0–72%), and wastewater (7–79%) in West and Central Africa [51]. Greater than 40% of wastewater from Tunisia were positive to ESBL-producing Enterobacteriaceae [52]. In this study, blaTEM-1 (n = 2) and blaCTX-M-55 (n = 3) were reported with MDR, which agreed with previous studies in aquatic environment and migratory birds [53, 54]. Furthermore, the blaTEM and blaCTX-M isolates were the common widespread genes from wild fish and aquatic environment [54, 55]. More specifically, the blaTEM-1, blaCTX-M-14 and blaCTX-M-15 genes were reported from marine bivalve mollusks [8]. Even though the low rates of ESBL producing bacteria were observed in this study, the positive ESBL isolates were commonly identified MDR bacteria. Hence, the occurrence of ESBL producing bacteria that harbored MDR signifies the public health threat.
The association between resistance to sulfamethoxazole and other predictors, including AMR, MDR, virulence genes, and ESBL production were examined under the logistic regression models (Tables 7, 8). The complexity of association among resistance and virulence of E. coli and Salmonella was observed. Sulfamethoxazole resistance in E. coli was positively associated with trimethoprim resistance, ESBL production, MDR, and the presence of addA1, strA, and sul3, but these isolates were negatively associated with lt, stx, and dfrA12. The major concern of these findings was almost half of E. coli carrying virulence genes were MDR bacteria. A co-selection of resistance and virulence can occur through mobile genetic elements such as integrons, transposons, and integrative conjugative elements [56]. The infection of resistant and virulent pathogens is detrimental to human health since they cause difficulty to treat and increase treatment failure. On the other hand, sulfamethoxazole resistance in Salmonella was positively correlated with resistance to ampicillin and trimethoprim, and invA, but they were negatively associated with ESBL production and stn. This finding indicated the complexity of AMR, virulence factors and resistance determinants in the environment. A quarter of Salmonella carrying virulence genes were MDR. Thus, sulfamethoxazole resistance isolates can co-selection to many classes of antimicrobials, virulence genes, and ESBL production. A previous study indicated that resistance and virulence plasmids were linked simultaneously [57]. As a result, the infection of resistant and virulent bacteria may cause more complicated treatment and increase morbidity and mortality rates due to failure of bacterial treatment.
Shiga toxin is bacterial exotoxin related to highly cytotoxic class II ribosome [58]. In this study, stx1, Shiga toxin-producing E. coli (STEC) was most frequently found in oysters and estuarine waters, while eae gene representing enteropathogenic E. coli (EPEC) was reported in estuarine water at a low rate (0.2%). A previous study indicated that none of virulence genes related to STEC and EPEC were identified in oysters and mussels from Atlantic Canada [29], in contrast to the results in this study. Wildlife and aquaculture, including fish and shellfish have been identified as one of important sources of STEC spillover from livestock animals [59]. The high rate of stx1 in this study raise public health concerns of seafood safety, since major clinical signs of STEC infection in humans are bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, and may be life-threatening.
The fimA, stn, and invA genes are common virulence genes that play an important role in the pathogenicity of Salmonella infection. The fimA gene is a common structural subunit of type 1 fimbrial protein, while stn is heat-labile Salmonella enterotoxin affecting epithelial cells [60, 61]. The invA gene is an important structural component of Salmonella pathogenicity island, which is related to invasion of gut epithelial tissues in human and animals [28]. In this study, 77.0% of Salmonella isolates were positive to invA gene, even though this gene has been used for confirmation of Salmonella in food animals. This agreed with previous studies where the absence of invA gene was found in poultry production [62, 63]. In seafood and environmental samples, some Salmonella isolates confirmed with biochemical test did not contain invA gene [64–66]. The absence of invA gene may be because Salmonella was not invasive or had other invasive mechanisms [67]. However, the absence of invA genes is a rare occasion. The combination of PCR and next generation sequencing (NGS) is proposed to increase sensitivity of Salmonella detection of resistance in environmental samples [68].
In conclusion, MDR and ESBL-producing E. coli are widespread in the estuarine environment, highlighting the need for continuing AMR monitoring programs in shellfish harvested area. Knowing the magnitude of AMR circulated in the environment can facilitate developing strategic action plans to mitigate the possible transmission of resistance bacteria among humans, animals, and environment. In addition to phenotypic detection of AMR, identification of AMR driving sources and monitoring of genetic information of resistance organisms are required to better understanding reduce the occurrence and transference of AMR in aquatic animals and estuarine waters. Oysters and estuarine water serve as overlooked natural reservoirs of AMR contamination. Awareness of seafood safety and increase personal hygiene are suggested to reduce AMR infection from seafood consumption.
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