Tetracycline Resistance in Salmonella spp. and Escherichia coli from Brazilian PoultryABSTRACT
Antimicrobial resistance (AMR) is a pressing global public health issue, driven by the widespread use of antibiotics in both human and veterinary medicine. Tetracyclines, widely used in livestock for growth promotion and disease treatment, have contributed significantly to the emergence of resistant bacterial strains. This study aimed to evaluate the tetracycline resistance profiles of Salmonella spp. and Escherichia coli isolates from the poultry supply chain in Brazil, and identify the primary tetracycline resistance genes involved. A total of 97 Salmonella strains and 195 E. coli strains were analyzed for tetracycline resistance using the disc diffusion method and PCR to detect resistance genes (tetA, tetB, tetC, tetD, tetE, and tetG). The results revealed high tetracycline resistance rates, with 88.66% of Salmonella strains carrying the tetA gene, 4.12% harboring tetB and 1.03% carrying tetD. Among E. coli isolates, 64.62% carried tetA, 65.13% carried tetB, and 43.59% exhibited both genes. The tetE gene was detected in 1.03% of E. coli strains. These findings highlight the widespread presence of tetracycline resistance determinants in poultry-associated bacteria, underscoring the need for stricter antimicrobial use regulations and enhanced AMR monitoring in animal production systems to mitigate public health risks.
Keywords:
Resistance; broiler; carcass; enterobacteria; foodborne pathogens; tetracycline
INTRODUCTION
Antimicrobial resistance (AMR) has emerged as a global public health concern, with implications for both human and animal health. It is primarily driven by antibiotic use in human and veterinary medicine and further exacerbated by the increased global movement of people, animals and food (World Health Organization, 2014). The widespread application of antimicrobials in animal production has been linked to the rise of resistant microorganisms, which can be transmitted to the environment (including soil, food, and aquatic ecosystems), thereby contributing to the emergence of resistant strains (Endale et al., 2023). The food chain, particularly through products such as milk, eggs, and especially meat, serves as a major route for the transmission of antimicrobial-resistant bacteria and resistance genes across different hosts (Calero et al., 2018; Caneschi et al., 2023). This has led to increasing recognition of the urgent need to prevent inappropriate antibiotic use and reduce antibiotic consumption in animal husbandry, aquaculture, and human medicine (World Health Organization, 2014).
Salmonella spp. and Escherichia coli are commonly present in the microbiota of poultry, which may represent a risk of transmitting pathogenic strains via the consumption of animal-derived products (CDC, 2019; EFSA, 2022). Multidrug-resistant Salmonella strains have been identified in food chain products, serving as potential sources of widespread dissemination and contributing to a global public health crisis. (Castellanos et al., 2018; Xu et al., 2020). Meanwhile, Escherichia coli, as commensal bacteria, are ubiquitous in both animals and humans. Due to their widespread presence, monitoring of commensal bacteria allows for the assessment of selective pressures across various populations, making them an essential tool for the early detection of emerging AMR in livestock and its potential transmission to animal-derived foods (EFSA, 2024).
Since their introduction in the 1950s, tetracyclines have been extensively used for their broad-spectrum efficacy and low toxicity, making them a common choice for treating infections in humans and animals, as well as for promoting growth in animal production systems. However, their extensive use in livestock to enhance growth rates has contributed to the development of tetracycline resistance in various bacterial species, leading to restrictions on their clinical use (Pavelquesi et al., 2021). Tetracycline resistance may arise from one or more factors, including the acquisition of mobile genetic elements carrying resistance genes, mutations in the ribosomal binding site, and/or chromosomal alterations leading to the upregulation of intrinsic resistance mechanisms. The mechanisms of resistance conferred by the acquisition of tet genes include efflux pumps, enzymatic inactivation of tetracyclines, and ribosomal protection (Grossman, 2016). Among these, the efflux pump mechanism is one of the most extensively studied in Salmonella and Escherichia coli due to its significant role in mediating resistance to tetracycline antibiotics (Gargano et al., 2021; Gharajalar, 2017). Representative tet genes associated with efflux pumps include tetA, tetB, tetC, tetD, tetE, and tetG (Chopra & Roberts, 2001).
Therefore, the objective of this study was to assess the tetracycline sensitivity profile of Salmonella spp. and E. coli isolates obtained from the poultry supply chain. In addition, it aimed to investigate the primary tetracycline resistance determinants associated with resistance to this class of antibiotics.
MATERIALS AND METHODS
A total of 97 Salmonella strains previously recovered from cloacal swabs (n=32) and broiler carcasses swabs (n=65) from slaughterhouses under Federal Inspection (Midwest, South, and Southeast regions of Brazil) between 2013 and 2019 were investigated. These strains belonged to the following serotypes: Schwarzengrund (n=2), Heidelberg (n=72), Minnesota (n=5), Saintpaul (n=3), Mbandaka (n=2), Agona (n=1), Senftenberg (n=2), Typhimurium (n=2) and non-typeable (n=8). Additionally, E. coli strains isolated from feces (n=91) and carcasses (n=104) from slaughterhouses operating under the State Inspection Service (Southeast region of Brazil) were also investigated.
Isolates were screened for resistance to tetracycline (30 μg) (Cefar Diagnostica, Brazil) by the disc diffusion method, as described in Clinical and Laboratory Standards Institute (CLSI 2023) guidelines. Approximately 108 CFU/ml (equivalent to a 0,5 McFarland turbidity standard) was inoculated onto Mueller-Hinton plates (Hi-Media, India) and incubated at 37°C for 18 to 20h. Escherichia coli strain ATCC 25922 was used as a control for the test.
The strains were tested for the tetracycline resistance genes tetA, tetB, tetC, tetD, tetE and tetG. A multiplex PCR (Ng et al., 2001) was performed in a final volume of 25 µL, containing 1× buffer, 0.2 µM of each dNTP, 0.2 µM of each primer, 1 U of Taq polymerase (Promega, Brazil), and 100µg of DNA. The PCR cycling conditions consisted of an initial denaturation at 95°C for 3 minutes, followed by 30 cycles of amplification (denaturation at 95°C for 15 seconds, annealing at 59°C for 15 seconds, and extension at 72°C for 20 seconds), with a final extension at 72°C for 5 minutes. The PCR products were separated on a 1.5% agarose gel submerged in Tris-Borate-EDTA (TBE) buffer, stained with 0.5 μg/mL of ethidium bromide (Ludwig Biotec, Brazil) and subjected to electrophoresis at 90 V for 40 min. Amplicons were visualized under ultraviolet light using a transilluminator, and images were captured using the L-Pix photo-documentation system (Loccus, Brazil).
DNA sequencing was conducted on the two samples that tested positive for the tetD and tetE genes. The amplicons were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Brazil) and subsequently sequenced on an ABI 3730XL DNA sequencer (Applied Biosystems, USA) at the Fiocruz Sequencing Platform. The resulting DNA sequences were deposited in GenBank under the accession numbers PV035088 and PV035089.
RESULTS
Table 1 demonstrates that all 25 Heidelberg strains and 3 Minnesota strains isolated from feces were resistant to tetracycline according to the disc diffusion method and were found to carry the tetA gene by PCR analysis. Additionally, all 4 non-typable strains were also resistant and carried the tetA gene. Among these, 1 strain carried only the tetA gene, while 3 strains carried both the tetA and tetB genes.
Distribution of Tetracycline Resistance and Associated Resistance Genes (tetA, tetB, tetC, tetD, tetE, tetG) in Salmonella Isolates from Feces and Carcass Sources.
In contrast, all Agona, Mbandaka, Seftrenberg, Typhimurium and non-typable strains isolated from carcasses were susceptible to tetracycline, as determined by the disc diffusion method. Among the 2 susceptible Mbandaka strains, 1 carried the tetA gene as detected by PCR analysis. Similarly, of the 4 susceptible non-typable strains, 1 also carried the tetA gene according to PCR analysis. All Minnesota and Saintpaul strains were resistant to tetracycline and carried the tetA gene, with 1 Saintpaul strain carrying both the tetA and tetB genes. Of the 47 Heidelberg strains analyzed, 46 were resistant to tetracycline and carried the tetA gene, whereas 1 of the 2 Schwarzengrund strains were resistant to tetracycline and carried the tetA and tetD genes.
Table 2 indicates that, out of the 91 strains isolated from feces, 89 were resistant to tetracycline according to the disc diffusion method. Among these, 66 strains were found to carry the tetA gene by PCR analysis, while 67 strains carried the tetB gene. Additionally, 47 strains were found to have both the tetA and tetB genes.
Distribution of Tetracycline Resistance and Associated Resistance Genes (tetA, tetB, tetC, tetD, tetE, tetG) in E. coli Isolates from Feces and Carcass Sources.
In comparison, out of the 104 strains isolated from carcasses, 90 were resistant to tetracycline as determined by the disc diffusion method. Of these, 60 strains carried the tetA gene by PCR analysis and 60 strains carried the tetB gene. Only 2 strains carried the tetE gene and 38 strains were found to have both the tetA and tetB genes.
The results demonstrate that tetracycline resistance is widely disseminated among the analyzed strains, with both resistant phenotypes and genotypes detected across all surveyed regions: Midwest, South, and Southeast of Brazil.
DISCUSSION
AMR is one of the major challenges of the century and the use of antimicrobials in livestock production has been associated with a rise in the prevalence of resistant microorganisms (Dias et al., 2021). Tetracyclines continue to be the most used class of antimicrobials in animal health worldwide (World Organisation for Animal Health, 2022). Despite the prohibition of tetracycline as a growth promoter in Brazil (Brazil, 2009), this antibiotic continues to be extensively used in veterinary medicine (Santana et al., 2023). This ongoing use may be associated with the high frequency of resistance observed in Salmonella and E. coli strains isolated from broiler carcasses and feces in the present study. Tetracyclines exhibit low oral bioavailability (Koutsoumanis et al., 2021), and the unabsorbed antibiotics can quickly alter the intestinal microbiota (Hansen et al., 2002), remaining microbiologically active in feces and impacting environmental microorganisms as well.
In this study, tetA was the most prevalent gene among the Salmonella strains, detected in 88.66% of the analyzed samples (86/97). The high prevalence of tetA has also been reported by other authors, indicating that this gene is widely distributed among Salmonella isolates of animal origin (Abd El-Aziz et al., 2021; Gargano et al., 2021; Penha Filho et al., 2023; Geyi et al., 2024). In contrast, the tetB gene was found in 4.12% of the strains (4/97), and tetD in 1.03% (1/97). These results are consistent with Gargano et al. (2021), who also reported low prevalence rates of tetB and tetD in Salmonella spp. isolates. Notably, some Salmonella isolates in our study carried more than one gene. Specifically, four isolates harbored both tetA and tetB, while one carried tetA and tetD. The latter combination was also reported by Gargano et al. (2021), whose findings, along with our data, underscore the co-existence of efflux-related resistance determinants within single strains. According to Pavelquesi et al. (2021), tetA and tetB are the most frequently detected tetracycline resistance genes in Salmonella spp., a pattern also observed in our study. Despite the absence of tetC, tetE, and tetG genes among the analyzed isolates, their presence in Salmonella strains has been reported in previous research (Adesiji et al., 2014; Gargano et al., 2021). These findings reinforce the idea that tetE is less likely to disseminate compared to other tet genes, as it is typically associated with large plasmids that are neither mobile nor conjugative (Roberts, 1994).
The prevalences of the tetA and tetB genes in the E. coli isolates were 64.62% (126/195) and 65.13% (127/195), respectively. These values are consistent with findings from other authors who also reported the predominance of these genes in poultry production isolates from different countries (Jahantigh et al., 2020; Racewicz et al., 2022; Alam et al., 2023). However, unlike those studies, where tetA was the dominant gene, our results indicate a predominance of tetB. Additionally, 43.59% (85/195) of the strains exhibited both resistance determinants. The discrepancies observed in comparison to other studies and the high prevalence of strains harboring both genes can be attributed to varying antimicrobial use practices in the analyzed regions, which may influence the frequency of resistance determinants such as tetA and tetB. Furthermore, these associations of resistance determinants are influenced by gene localization, as co-localization on specific plasmids can lead to distinct patterns of co-selection based on the use of different antimicrobial agents (Lanz et al., 2003). Moreover, 1.03% (2/195) of the strains were identified as possessing the tetE gene. This gene has also been documented by Gharajalar (2017) in E. coli isolates from poultry, and is reported less frequently than other tetracycline resistance determinants such as tetA and tetB. Although none of the isolates in this study harbored the tetC, tetD, or tetG genes, the presence of tetC and tetD has been reported in E. coli strains by Jahantigh et al. (2020), while tetG was also described by Gharajalar (2017).
The detection of tetracycline-resistant phenotypes and associated resistance genes across isolates from all surveyed regions of Brazil (Midwest, South, and Southeast) highlights the broad dissemination of antimicrobial resistance within the national poultry production system. These findings are consistent with previous studies reporting widespread tetracycline resistance in poultry-associated bacteria in Brazil (Rabello et al., 2020; Vásquez-Jaramillo et al., 2023), reinforcing concerns about the rising antimicrobial resistance to this class of antibiotics, which continues to be used against a broad spectrum of pathogens, including Gram-negative bacteria (Pearson et al., 2025).
CONCLUSION
In this study, tetracycline-resistant strains were found among Salmonella spp. and E. coli isolates obtained from the poultry supply chain in the Midwest, South, and Southeast regions of Brazil. The tetracycline resistance determinants identified in Salmonella spp. strains were tetA, tetB, and tetD, while those found in E. coli strains were tetA, tetB, and tetE. Therefore, considering the risk that AMR in bacteria isolated from the food production chain poses to both animal welfare and public health, it is essential to implement stricter control measures regarding the use of antimicrobials in animal production, as well as to establish robust monitoring systems that track the development of this issue over time.
ACKNOWLEDGEMENTS
None.
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FUNDING
This study was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (E-26/200.028/2024 and E-26/200.029/2024) and Federal Fluminense University.
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DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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DISCLAIMER/PUBLISHER’S NOTE
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
