Selman Bahadır Orhan
Ali Anil Suleymanoglu
Ali Aydin- Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Türkiye
Objectives: Salmonella, a major foodborne pathogen, is a primary concern due to its role in spreading antibiotic resistance. Raw chicken meat samples (n = 210) were collected from various retail locations in Istanbul.
Methods: The food samples were isolated according to ISO 6579-1 and 13 (6.2%) of Salmonella strains confirmed through PCR, agglutination tests, and Sanger sequencing; S. Infantis (84.6%) was identified as the dominant type. The other types found included S. Enteritidis (7.7%) and S. Virchow (7.7%). Additionally, antibiotic susceptibility was tested according to EUCAST and CLSI standards in different Salmonella serotypes. The serotypes were analyzed for susceptibility to 13 antibiotics using agar-disk diffusion assays, and resistance levels were determined via E-test.
Results: The disc diffusion method revealed resistance to cefazolin across all Salmonella serotypes. High resistance rates were also observed for pefloxacin (84.6%), azithromycin (76.9%), and tetracycline (84.6%). Multidrug resistance was identified in 11 (84.6%) strains by the disc diffusion test. The minimum inhibitory concentration testing with MIC test strips showed high tetracycline resistance at 84.6%. The blaTEM gene was found in 30.7% of strains, while blaCTX-M subgroup 1 (7.7%) and blaCTX-M subgroup 9 (30%) were detected by multiplex PCR; however, and blaCTX-M, blaOXA-2, and blaSHV genes were not present. Resistance to carbapenem and colistin was also checked via PCR, and blaOXA-48, blaVIM, blaNDM, blaKPC, and mcr genes were not detected in the Salmonella serotypes.
Conclusion: This pioneering study provides a comprehensive analysis of serotyping and ESBL production in Salmonella strains isolated from Istanbul.
1 Introduction
Salmonella (S.), a major foodborne pathogen, is a leading cause of diarrheal diseases worldwide. It can be present at all stages of the food supply chain and may contaminate food products through cross-contamination. Furthermore, the development of antibiotic resistance in Salmonella, which can endure various conditions, represents a significant and urgent public health threat. The rise of antimicrobial resistance makes it increasingly difficult to treat salmonellosis symptoms. Food products such as poultry, meat, eggs, and milk (1, 2) are primary sources of salmonellosis in humans. However, poultry remains Salmonella’s main reservoir, posing a threat to food safety through eggs and chicken meat (3, 4).
Chicken meat is one of the world’s most popular and preferred animal protein sources. Poultry is also the second most produced and consumed meat in Europe, with remarkable consumption in countries such as England, France, Spain, Ireland, and Portugal (5). However, Türkiye is the world’s seventh-largest exporter, with poultry meat production expected to reach 2,245,770 tons in 2021 (6). Indeed, worldwide production and consumption of fresh poultry meat are projected to increase significantly in 2024, with a 3.5% increase compared to poultry production and consumption in 2023 (7). Nevertheless, according to the “One Health 2021 Zoonoses Report” of EFSA and ECDC (8), the prevalence of Salmonella was reported as 7.3% in fresh poultry meat tested and offered for retail sale and 7.6% in meat products made from poultry meat and intended for consumption, cooked. On the other hand, the distribution of Salmonella serotypes in chicken meat has changed compared to previous studies (3, 9).
Antibiotic resistance increasingly threatens human and animal health. Due to the uncontrolled and overuse of antibiotics, microorganisms resistant to various antibiotics are identified daily. The World Health Organisation (WHO) states that antibiotic resistance is one of the most critical threats to humanity (10). Enterobacterales, including Salmonella, are a significant public health threat due to the development of antibiotic resistance today (11). The WHO ranks pathogenic bacteria for public health according to the importance of antibiotic resistance, and fluoroquinolone-resistant Salmonella is in the high-importance group (12). Moreover, Salmonella spp., Campylobacter spp., indicator Escherichia coli (E. coli), and methicillin-resistant Staphylococcus aureus are among the antibiotic-resistant zoonotic and indicator bacteria of food, human, and animal origin that pose a threat in the European Union (EU) (13). Various antibiotic-resistant Salmonella serotypes have been reported by many researchers (14–16).
ESBL-producing Enterobacterales present a major global public health threat. Last-resort antibiotics like carbapenems and colistin are used to fight this, especially when there’s resistance to 3rd-generation cephalosporins. ESBLs make antibiotics such as penicillins, monobactams, and cephalosporins ineffective, which are commonly used to treat diseases in humans and animals. They can also break own carbapenem antibiotics, though the effectiveness of carbapenems is evaluated separately. Carbapenem-resistant Enterobacterales and those resistant to 3rd-generation cephalosporins are classified as critical pathogens by WHO (12). Consequently, colistin—one of the few remaining treatment options, particularly against carbapenem-resistant bacteria—becomes a vital focus (11).
The aim of this study was (a) to examine the presence of Salmonella in chicken meat samples collected in the Asian and European parts of Istanbul with conventional methods and PCR; (b) serotyping of Salmonella isolates by sanger sequence and detection of phylogenetic affinities; (c) to determine the antibiotic susceptibility against 9 antibiotics groups (aminoglycosides, cephalosporins, macrolides, fluoroquinolones, penicillin, phenicol, sulfonamides, tetracyclines and carbapenems) by agar-disc diffusion assays according to the EUCAST and CLSI standards and detection Minimum Inhibitory Concentration (MIC) value of resistant Salmonella serotypes using E-test, and (d) to investigate phenotypic ESBL production and the ESBLs genes (blaTEM, blaCTX-M subgroup (1–2–8-9-25/26), blaSHV and blaOXA-2), the carbapenem (blaOXA-48, blaVIM, blaNDM and blaKPC) and mobilized colistin (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) as resistance genes in Salmonella serotypes by PCR.
2 Materials and methods
2.1 Sampling
A total of 210 raw chicken meat samples were collected from different sale points in Istanbul, Türkiye, between May and August 2021. The samples from the European part of Istanbul included drumsticks (n = 15), breasts (n = 25), thighs (n = 26), and wings (n = 40). From the Asian part, the samples consisted of drumsticks (n = 15), breasts (n = 33), thighs (n = 27), and wings (n = 29). All samples were transported under cold conditions (≤ + 4 °C) in thermal boxes to the Department of Food Hygiene and Technology at Istanbul University-Cerrahpasa immediately after collection.
2.2 Isolation and identification of Salmonella by conventional methods
Salmonella was isolated and identified according to the ISO 6579-1 standard method (17). 25 g of the collected raw chicken meat samples and 225 mL of buffer peptone water (Oxoid CM 0509, Basingstoke, United Kingdom) were added and homogenized in a stomacher (Interscience, Saint Nom la Bretèche, France). Subsequently, 1 mL of inoculum was transferred into 10 mL Mueller Kaufmann Tetrathionate Broth (MKTTn) (Oxoid, CM1048), and 0,1 mL of inoculum from stomacher bags was transferred to 10 mL Rappaport-Vassiliadis Broth (Oxoid, CM0866). The MKTTn and RVS broth were incubated at 37 °C and 41.5 °C for 24 ± 3 h, respectively. Subsequently, the samples were inoculated on Xylose Lysine Desoxycholate (Oxoid, CM0469) and Hectoen Enteric Agar (Oxoid, CM0419) and incubated at 37 °C for 24 h. Suspected Salmonella isolates were inoculated on Nutrient agar (Millipore, 105,450) for purification and incubated at 37 °C for 24 h. The purified colonies were stored in Tryptone Soya Broth (TSB) (Oxoid, CM0129) with 20% glycerol at −18 °C.
For biochemical confirmation, suspected Salmonella colonies were transferred to Triple Sugar Iron Agar (Oxoid, CM0277), Lysine Iron Agar (Oxoid, CM0381), and Urea Broth (Oxoid, CM0071) media and incubated at 37 °C for 24 h (17). Subsequently, isolated Salmonella spp. were confirmed by PCR, agglutination test, and Sanger sequencing.
In the analysis, S. typhimurium ATCC 14028, S. Enteritidis ATCC 13076, and S. Infantis ATCC 51741 were used as positive controls, and while E. coli ATCC 25922 was used as a negative control.
2.3 Verification of Salmonella isolates by PCR
2.3.1 DNA extraction
The DNA extraction method of was used to extract genomic DNA of suspected Salmonella isolates according to Liu et al. (18). Epoch2 (BioTek, United States) tested the acquired DNA for quality and stored at −20 °C.
2.3.2 Confirmation of Salmonella isolates by PCR
The identification of Salmonella spp. was performed using PCR. invA F-(5′- GTGAAATTATCGCCACGTTCGGGCAA-3′) and invA R-(5′- TCATCGCAC-CGTCAAAGGAACC-3′) (284 bp), which are specific primers for invA (Salmonella-specific gene) were used in PCR. The PCR assay was conducted with the following conditions: initial denaturation at 72 °C for 7 min, 35 cycles of 94 °C for 60 s, 53 °C for 120 s, and 72 °C for 180 s. Following the last cycle, there was a 7 min incubation at 72 °C (19). Subsequently, the PCR products were resolved on 1% (w/v) agarose gels in 1 × Tris-acetate-EDTA (TAE) buffer. The bands in the agarose gels were visualized using the SafeView™ Classic stain (ABM, Richmond, BC, Canada) in the Infinity Gel Imaging System (Vilber Lourmat, Marne-la-Vallée, France).
2.3.3 Detection of some non-typhoidal Salmonella serotypes by mPCR
The mPCR method was used to detect some of the non-typhoidal Salmonella types. It was aimed to identify S. Enteritidis-specific SdfI; S. typhimurium-specific fliC-i; S. Dublin-specific fliC-gp, and S. Stanleyville-specific fliC-z4,z23 gene regions (Table 1).
Table 1. Primers for detection of non-typhoidal Salmonella types by mPCR.
The mPCR master mix was made according to Tennant et al. (20). The cycling parameters of the mPCR involved denaturation at 95 °C for 2 min, followed by 25 cycles comprised of heating to 95 °C for 30 s, 64 °C for 30 s, and 72 °C for 15 s, and a final step of 72 °C for 5 min.
2.3.4 Agglutination test for investigation of Salmonella serovars
Serovars of Salmonella isolates were confirmed using the slide agglutination test by identifying the types of the O (SSI Diagnostica, Denmark) and H (SSI Diagnostica, Denmark) antigens with diagnostic sera for Salmonella according to the Kauffmann-White scheme (21). The test was conducted at the National Food Reference Laboratory of the Ministry of Agriculture and Forestry of the Republic of Türkiye and the National Enteric Pathogens Reference Laboratory of the General Directorate of Public Health of the Ministry of Health of the Republic of Türkiye.
2.3.5 Sanger sequencing
Following genomic DNA extraction, amplification was performed using the forward 5’-AGAGTTTGATCCTGGCTCAG-3′ and reverse 5’-CTACGGCTACCTTGTTACGA-3′ primers (22) specific to the 16S rRNA region, which were used to amplify an approximately 1,500-base gene region by PCR. Readings obtained from Sanger sequencing primers were assembled into a contig to create a consensus sequence.
The CAP contig assembly algorithm in BioEdit software was used for this process. Sequence analysis results were evaluated using BLAST analysis and similarity scores obtained from NCBI GenBank. Sequence analysis results were evaluated using BLAST analysis and similarity scores obtained from NCBI GenBank. These findings gave clues about the phylogenetic relationships of the samples studied. Additionally, the high similarity scores indicated a close genetic relationship with known species, highlighting the potential for further research in this area.
2.3.6 Phylogenetic tree construction and bioinformatics analysis
After sequencing, 16S rRNA sequences were used for homology search and phylogenetic analysis using NCBI-BLAST.1 The Phylogenetic Tree for the sequences of the respective strains was constructed using MEGA-11 Software (Figure 1).
Figure 1. Sanger sequence-based phylogenetic tree of Salmonella enterica strains from raw chicken meat.
2.4 Antibiotic susceptibility tests in Salmonella serotypes
2.4.1 Screening for antibiotic susceptibility using disc diffusion tests
The antibiotic susceptibility of the isolated Salmonella serotypes was tested using agar disc diffusion on Mueller–Hinton Agar (MHA; Oxoid CM 337), in accordance with the European Committee on Antimicrobial Susceptibility Testing (23). Results were interpreted following EUCAST (24) and CLSI (25) guidelines. The disc diffusion test was performed on MHA for 13 different antibiotics (Oxoid, Basingstoke, United Kingdom): kanamycin (CT0026B, 30 μg), gentamicin (CT0024B, 10 μg), cefotaxime (CT0166B, 30 μg), cefazolin (CT0011B, 30 μg), azithromycin (CT0906B, 15 μg), ciprofloxacin (CT0425B, 5 μg), pefloxacin (CT0661B, 5 μg), ampicillin (CT0003B, 10 μg), amoxicillin-clavulanic acid (CT0223B, 30 μg), chloramphenicol (CT0013B, 30 μg), trimethoprim/sulfamethoxazole (CT0052B, 1.25/25 μg), tetracycline (CT0054B, 30 μg), and imipenem (CT0455B, 10 μg). Escherichia coli ATCC 25922 served as the quality control strain. Petri dishes were examined after 18 ± 2 h of incubation at 37 °C, and Salmonella serotypes were categorized as sensitive or resistant based on zone diameter breakpoints for each antimicrobial, according to EUCAST (24) and CLSI (25) guidelines. For tetracyclines and azithromycin, only CLSI (25) provided breakpoints, while EUCAST (24) did not.
2.4.2 Detection of minimum inhibitory concentration in resistant Salmonella serotypes
E-test strips were used to measure the minimum inhibitory concentration of Salmonella serotypes according to the EUCAST (24) and CLSI (25) breakpoint tables; only the antibiotic resistance was determined by a disk diffusion test. Accordingly, test strips were used to cefazolin (Liofilchem, 92,174), pefloxacin (Liofilchem, 92,041), azithromycin (Liofilchem, 92,030), ciprofloxacin (Liofilchem, 920,451), trimethoprim/sulphamethoxazole (Liofilchem, 92,123), kanamycin (Liofilchem, 92,034), chloramphenicol (Liofilchem, 92,075), tetracycline (Liofilchem, 92,114), cefotaxime (Liofilchem, 92,007), and amoxicillin-clavulanic acid (Liofilchem, 92,024).
2.4.3 Double disk synergy test for phenotypic confirmation of ESBL production
Double disc synergy test (DDST) was used for ESBL phenotypic confirmation test. CAZ Oxoid, CTO412B, 30 μg, CTX, and AMC antibiotic disks were plated 20 mm apart on MHA with the AMC disc in the middle. The plates were incubated at 37 °C for 24 h (26).
2.4.4 Investigation of ESBLs genes by PCR
The PCR assay was conducted to determine whether the isolates have blaSHV, blaTEM, blaCTX-M, and blaOXA. PCR mix was as follows (final 25 μL): 2.5 μL DNA samples, 10X KCL buffer 2.5 μL, dNTP mix 2.5 μL, MgCl2 1.5 μL, each primer 0.5 μL, Taq DNA polymerase (Thermo Fisher EP0404, United States) 0,4 μL and dH2O 12 μL. mPCR to detect ESBL’s genes condition follows initial denaturation at 95 °C for 15 min, followed by 30 cycles of 94 °C for 30 s, 62 °C for 90 s, and 72 °C for 60 s, and with a final extension at 72 °C for 10 min in the thermal cycle (Applied Biosystems, Veriti, USA) (27). The amplified PCR products were subjected to electrophoresis at a 1.5% agarose gel with a 5 μL safe view (Abm, Richmond, Canada) (Table 2). Klebsiella pneumoniae NCTC 13443 metallo beta-lactamase NDM-1 was used as a reference strain.
Table 2. Primers and target genes detection of ESBLs and carbapenem-resistance genes by mPCR.
The PCR assay was conducted to determine whether the isolates have blaCTX-M subgroup (1–2-8, 25/26, 9) (28). The amplified PCR products were subjected to electrophoresis at a 1.5% agarose gel with a 5 μL safe view (Abm, Richmond, Canada) (Table 2).
2.5 Investigation of carbapenem resistance genes by PCR
Carbapenem resistance genes (blaOXA-48, blaVIM, blaNDM, blaKPC) were investigated using monoplex PCR. The PCR mix was as follows (final 25 μL): 3 μL DNA samples, 10X KCL buffer 2.5 μL, dNTP mix (dATP, dCTP, dGTP, and dTTP) 2.5 μL, MgCl2 1.5 μL, each primer 0.5 μL, Taq DNA polymerase (Thermo Scientific, United States) 0.14 μL, and dH2O 12 μL. Klebsiella pneumoniae NCTC 13443 metallo beta-lactamase NDM-1 was used as a reference strain.
The PCR assay conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, melting temperature found for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 10 min in the thermal cycler (Applied Biosystems, Veriti, USA). Amplificons were subjected to electrophoresis at a 1.5% agarose gel (w/v) containing 5 μL SafeView (Abm, Richmond, Canada) (29) (Table 2).
2.6 Investigation of mobilized colistin resistance genes by mPCR
The PCR assay investigated whether the Salmonella serotypes (n = 13) contained mobilized colistin resistance genes (Table 3). Conditions of multiplex PCR to detect mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were as follows: initial denaturation at 95 °C for 15 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 90 s, and 72 °C for 60 s; and a final extension at 72 °C for 10 min in the thermal cycler (29). E. coli NCTC 13846 was used as the reference strain. Amplification products were analyzed in 1.5% (w/v) agarose gel containing 5 μL SafeView.
Table 3. Primers to detect different mobilized colistin resistance genes (1–5) by mPCR.
2.7 Statistical analysis
Data on the prevalence of Salmonella in chicken parts were analyzed using Pearson’s chi-square and Fisher’s exact tests for independence to determine whether Salmonella prevalence differed across types and regions in Istanbul, Türkiye (α = 0.05).
3 Results
3.1 Detection of Salmonella in chicken meat samples in Istanbul and phylogenetic relationship
In this study, 14 raw chicken meat samples were found to be positive for Salmonella isolates analyzed using the conventional ISO method (17). In total, 92.9% (13/14) of Salmonella isolates confirmed by PCR. 1 isolate was identified as S. Enteritidis by mPCR, but the serotype of the other Salmonella isolates could not be determined. Therefore, the agglutination test and Sanger sequencing methods were preferred for molecular confirmation. These Salmonella 13 isolates were serotyped by Sanger sequencing, and S. Infantis 11/13 (84.6%) was the dominant serotype. In addition, one (7.7%) Salmonella isolate serotyped as S. Virchow, and the other (7.7%) Salmonella strains were S. Enteritidis (Table 4).
Table 4. Distribution of Salmonella serotypes in chicken parts from the Asian and European sides of Istanbul.
The S. Infantis strain obtained from the Asian side and the S. Virchow strain obtained from the European side were phylogenetically very close (Figure 1).
3.2 Antibiotic susceptibility tests in Salmonella serotypes
In this study, a disc diffusion test was performed to assess antibiotic susceptibility in Salmonella strains. 13 Salmonella serotypes were resistant to cefazolin, and 92.3 and 84.6% were resistant to pefloxacin (a fluoroquinolone) and tetracycline, respectively, according to the CLSI (25). However, all strains were susceptible to ampicillin, imipenem, and gentamicin by EUCAST (24) and CLSI (25) guidelines (Table 5).
Table 5. Clinical and laboratory standards institute (CLSI) and the European committee on antimicrobial susceptibility testing (EUCAST) as assessed by the disc diffusion method of Salmonella serotypes (n = 13) [Resistant (“R”); or susceptible (“S”)].
3.3 Detection of MIC in resistant Salmonella serotypes
Salmonella strains demonstrating antibiotic resistance in the disc diffusion assay were selected for E-Test testing to determine MICs. Based on MIC results, the highest tetracycline resistance among Salmonella strains was 84.6%. Resistance to tetracycline is followed by resistance to macrolides (53.8%) and aminoglycosides (38.4%). Interestingly, the disc diffusion test showed that all strains were resistant to cefazolin; however, the MIC test did not support this (Table 6).
Table 6. The MIC, as assessed by E-test, for 10 antimicrobial agents against Salmonella serotypes.
The present study detected MDR in 72.7% of S. Infantis strains. However, MDR was not determined in S. Enteritidis and S. Virchow strains. MIC values were prioritized in the MDR detected. 75% (6/8), 12.5% (1/8), and 12.5% (1/8) MDR S. Infantis strains were isolated from the wing, drumstick, and thigh, respectively. Additionally, MDR S. Infantis strains were equally distributed in the European and Asian sides of Istanbul (Table 7).
Table 7. Antibiotic resistance profiles of Salmonella strains.
3.4 ESBL-producing Salmonella, carbapenem, and colistin resistance in Salmonella serotypes
As a result of DDST used for phenotypic confirmation of ESBL production, 38% of isolates were found to produce ESBLs. As a result of this study, 30.7% of Salmonella serotypes containing the blaTEM gene by PCR, blaOXA-2, and blaSHV were not detected. These strains were S. Infantis (2), S. Virchow, and S. Enteritidis. As a result of the examination of resistance genes related to the blaCTX-M subgroups (1, 2, 8, 9, and 25/26) by mPCR, the blaCTX-M gene of subgroup 1 was detected in 1 strain (7.7%), while the gene of subgroup 9 was identified in 4 strains (30%). PCR investigated carbapenem and colistin resistance in Salmonella serotypes, and the presence of blaOXA-48, blaVIM, blaNDM, blaKPC, mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes was analyzed. Carbapenem resistance using the disc diffusion method with imipenem also investigated. In our study, no Salmonella serotypes were included in the relevant genes, carbapenem and colistin resistance were not detected in any strains. Additionally, imipenem-resistant Salmonella strains were also not determined.
4 Discussion
The worldwide consumption of chicken meat is relatively high due to economic, religious reasons, and the popularity of chicken products. As a result, chicken meat is favored in many food businesses and restaurants (30). A total of 6.2% (13/210) of Salmonella strains were isolated from chicken meat samples: the wing (76.9%; 10/13), thigh (15.3%; 2/13), and drumstick (7.7%; 1/13). Salmonella was not found in any breast samples in this study. Similarly, Sezen et al. (31) reported 6% of Salmonella spp. in chicken meat samples from Istanbul. In another study, 3% of Salmonella was detected in chicken meat samples, with 1% found in wings and drumsticks in Türkiye (32). A study in Brazil found 46.1% (53/115) of Salmonella spp. on chilled chicken meat collected at retail points (33). Perin et al. (34) also reported 31.5% Salmonella in chicken meat samples from Brazil. Conversely, Pavelquesi et al. (33) and Perin et al. (34) observed higher rates of Salmonella in chicken wings compared to other parts. Given that both Brazil and Türkiye are large chicken meat producers, understanding Salmonella epidemiology in these countries is important. The differences in Salmonella prevalence reported across studies may also result from variations in detection methods (35).
A previous study reported Salmonella spp. in 15% of 100 raw chicken meat samples collected from Istanbul, with S. Enteritidis making up a relatively high proportion (26.6%) of the isolates (30). The findings of our study, along with those of reference (30), provide important insights into Salmonella isolation and identification in similar samples from the same province. Additionally, the number of Salmonella serotypes originating from chicken meat was higher on the European side of Istanbul (53.9%; 7/13) compared to the Asian side (46.1%; 6/13). Similarly, another study found that Salmonella prevalence in raw chicken carcasses was higher on the European side of Istanbul (53.3%; 8/15) (30). This difference may be due to the increasing population density on the European side and inadequate hygiene practices at points of sale.
Rapid molecular methods for detecting Salmonella include PCR-based assays and next-generation sequencing (NGS) (36). In a study, 21.2% of Salmonella strains were detected in raw chicken meat (24/113) collected from markets in Korea. Through of molecular analysis (MLST), they identified S. Enteritidis as the dominant strain (45.8%), followed by S. Virchow (25%), S. Montevideo (8.3%), S. Bsilla (8.3%), S. Bareilly (4.2%), S. Dessau (4.2%), and S. Albany (4.2%) (9). Previously, many researchers also reported S. Enteritidis as the most common species in chicken products (30, 35, 37, 38). However, in the present study, S. Infantis was identified as the dominant species. Similarly, recent studies have noted an increased prevalence of serotypes such as S. Infantis (3, 15, 39). Since 2011, S. Infantis has been reported as the fourth leading cause of Salmonellosis in humans (40). Additionally, S. Infantis was among the most frequently identified Salmonella species in the EU, accounting for 36.5% in chicken and 36.5% in other animal meat (41). These European findings are consistent with our study results. Moreover, it is noteworthy that Salmonella spp. (6.2%) was close to European data (7.3%) (8) and species-level data in our study. Although S. Infantis does not typically infect poultry, it remains a public health concern because this serotype can cause disease in humans (42).
The S. Infantis strain sourced from the Asian region and the S. Virchow strain sourced from the European region exhibited significant phylogenetic proximity. Starting in summer 2017, the S. Virchow outbreak continued in EU countries, and S. Virchow was monitored as a public health threat to poultry farms. As a result of the WGS analysis performed on this strain, it was reported that chicken meat-origin strains were predominant. Furthermore, comparing representative outbreak strains with existing S. Virchow ST16 genome profiles from non-human isolates showed that most matching isolates originated from broilers in Germany, the Netherlands, and France. Most human cases have been reported to have originated at local kebab restaurants. The hospitalization rate for cases caused by this agent was 38.5% in Germany, underscoring the importance of monitoring chicken meat throughout the food chain (43). Similarly, there was a very close genetic affinity between the ESBL-producing S. Enteritidis strain isolated from the Asian side and the S. Infantis strain isolated from the European side. The other ESBL-producing strain, S. Infantis (S20), was phylogenetically more distant from other ESBL-producing strains.
Antibiotic resistance is a significant public health concern because it can spread through food, from animals to humans, or via cross-contamination between different sources (11). Salmonella is one of the most important antibiotic-resistant zoonotic pathogens and serves as an indicator bacterium of food, human, and animal origin (13). In this study, the disc diffusion method was used to determine the antibiotic susceptibility of Salmonella strains. All isolates were resistant to cefazolin, while 92.3 and 84.6% showed resistance to pefloxacin (a fluoroquinolone) and tetracycline, respectively, according to CLSI guidelines (25). By contrast, all strains were susceptible to ampicillin, imipenem, and gentamicin, based on EUCAST (24) and CLSI (25) standards.
Comparable findings have been reported elsewhere. For instance, high resistance rates to trimethoprim–sulfamethoxazole were found in Salmonella strains isolated from chicken meat, with 61.2% in Iran (35) and 71.4% in Türkiye (44). Another study documented complete resistance to amoxicillin (100%). In comparison, resistance to cefotaxime (55.1%) and chloramphenicol (42.4%) was moderate, and no resistance to kanamycin (0%) was observed, contrasting with the resistance profile seen in our study (16). Pavelquesi et al. (33) found resistance to amoxicillin/clavulanic acid (83.3%), sulfonamides (64.1%), tetracycline (46.2%), and ciprofloxacin (65.4%) in Salmonella isolates from chicken meat in Brazil, using disc diffusion tests. Our study reported high tetracycline resistance (84.6%) in Salmonella strains, with comparable rates of fluoroquinolone resistance similar to Wang et al. (45). Conversely, tetracycline, amoxicillin-clavulanic acid, and ciprofloxacin resistance in E. coli (Enterobacterales) from chicken meat in Istanbul were 74.2, 87.1, and 45.5%, respectively (46). It is important to note that both studies were conducted in Istanbul within the same year, which is essential when monitoring antibiotic-resistant bacteria in chicken meat.
Rincón-Gamboa et al. (47) reported that tetracycline resistance was the most frequently observed through MIC tests. Additionally, researchers noted high resistance to cephalosporins and ampicillin, as determined by MIC. It is beneficial for public health that cephalosporin resistance, which is closely associated with extended-spectrum beta-lactamase, was not detected in the current study. Conversely, Rincón-Gamboa et al. (47) identified different serotypes of S. Infantis in most of the studies they examined, which aligns with our findings. Furthermore, Rau et al. (48) analyzed Salmonella in poultry meat in Brazil during 2014 and 2017 and found evidence of antibiotic resistance, particularly in Salmonella ser. Heidelberg and Salmonella ser. Minnesota. Resistance rates in 2014 and 2017 to cefotaxime (76/146, 52.1% and 124/163, 76.1%), ciprofloxacin (83/146, 56.9% and 145/163, 89.0%), and tetracycline (88/146, 60.3% and 135/163, 82.8%) were also identified by MIC testing. While tetracycline resistance was similar across studies, there was a significant difference in cephalosporin resistance rates compared to our results.
Multi-drug-resistant Enterobacterales constitute a significant health problem that threatens global health and is becoming increasingly difficult to treat. MDR-containing foodborne pathogens are instrumental in spreading antibiotic resistance and resistance from farm animals to the general public (11). The present study detected MDR in 72.7% of S. Infantis strains. Lee et al. (49) isolated S. Kentucky (25.58%), S. Reading (18.60%), S. Infantis (11.63%), and S. typhimurium (9.30%) in their study on 958 retail meat samples in the United States. 13.95% of the strains (n = 6) were found to contain MDR, and the distribution of these strains were S. Infantis (n = 4), S. Reading (n = 1), and S. Kentucky (n = 1). Similar to our results, tetracycline (52.17%) was the most resistant group in the MDR Salmonella strains isolated from chicken meat. Conversely, Lee et al. (49) reported that all strains were susceptible to azithromycin. Moreover, they noted that one S. Kentucky strain was resistant to amoxicillin-clavulanic acid, cefoxitin, and ceftriaxone. The fact that cephalosporin resistance, which is associated with ESBL production and is a significant issue in MDR, was not found in the present study is valuable for public health.
Chen et al. (50) found that S. Corvallis, S. Kentucky, and S. Agona were the dominant species among Salmonella isolates from pork, duck, and chicken meat offered for retail sale in South China. 80.1% of the Salmonella strains contained MDR. In addition, they stated that the MDR rate was incredibly high (91.8%) in S. Kentucky strains. Moreover, tetracycline (93.8%) was reported as the prominent antimicrobial agent. On the other hand, Wang et al. (51) reported that S. typhimurium (23.73%, 8,397/35,382) is the most prevalent serovar in both human and non-human sources in China, followed by S. Enteritidis, S. Derby, S. London, and S. Thompson. Researchers have identified ESBL-resistance, mobile colistin resistance, fosfomycin resistance, and mobile tigecycline resistance genes in MDR Salmonella strains (>45%). Another study found an MDR rate of 53.8% among Salmonella strains isolated from chilled chicken meat in Brazil (33). The most common antibiotic resistance rates were determined against amoxicillin/clavulanic acid (83.3%), followed by sulfonamide (64.1%) and tetracycline (46.2%) in MDR Salmonella strains. It has been observed that different serotypes are common in Salmonella strains from poultry meat reported from other continents, such as Asia (52, 53) and Brazil (34), and Europe (43), and that MDR to different antibiotics is higher in parallel. Researchers reported mainly tetracycline and sulfanamide resistance in Salmonella serotypes (33, 35, 43, 47). On the other hand, it is essential to note that cephalosporin resistance detected in ESBL-producing Salmonella strains was not observed in the present study, unlike other studies. Indeed, Aydin et al. (46) detected 79.2% MDR E. coli in raw chicken meat obtained from Istanbul in the same year as this study. They reported high resistance rates to cephalosporins, penicillins, and sulfonamides, although similar tetracycline resistance was also detected. Therefore, the characteristics of MDR Salmonella need to be investigated in more comprehensive studies.
As a result of DDST used for phenotypic confirmation of ESBL production, 38% of isolates were found to produce ESBLs. Kahraman et al. (54) reported a 37.5% rate of phenotypic ESBL production in Salmonella strains isolated from chicken carcasses collected in Istanbul. Unlike the present study, they did not detect any blaCTX-M genes. Notably, there is an approximate 7-year gap between the two studies’ sample collection periods. These findings suggest a rising trend in antimicrobial resistance over time, likely driven by continued antibiotic use. Additionally, the potential for antibiotic resistance conferred by mobilized genetic elements, such as plasmids, is a concern. This suggests that, while resistance rates may currently be undetectable, they could increase in the future. In this context, Aydin et al. (46) reported a high rate of ampicillin resistance (78.2%) and the presence of the blaTEM gene associated with this resistance (97.02%). Suleymanoglu et al. (55) reported a similar result for the blaTEM gene in all E. coli isolates, detecting 80% ampicillin resistance. The mobility of these genes on transposons and plasmids could further accelerate their spread and thus pose a potential threat to human health (44). Li et al. (56) reported that ampicillin resistance was 97.5% and that blaTEM was detected in 100% of 40 Salmonella strains isolated from 200 chicken carcasses in China. They detected a very high rate of ESBL-producing Salmonella. Kang et al. (57) isolated a 1.4% strain from 555 chicken meat samples in South Korea. These strains were identified as S. Enteritidis and differed from this study by carrying the blaCTX-M-1 gene and ampicillin resistance. Although no ampicillin resistance in Salmonella serotypes was observed in this study, the detection of the blaTEM gene at a 30% rate indicates that plasmid-mediated resistance has a complex epidemiology.
Wang et al. (51) reported that 59.9% of ESBL-producing Salmonella strains were CTX-M type beta-lactamases. Another study, Adigüzel et al. (58), conducted in 2016, found that 29.9% of Salmonella strains from retail chicken meat in Türkiye produced ESBL. They investigated the same blaCTX-M subgroups as this study and found that the blaCTX-M-8-25 gene was dominant. Although both studies were conducted in the same country, they took place in different regions, which may have contributed to the differences in blaCTX-M subgroup. Additionally, there is about a 5 year gap between the sample collection years of the two studies, and it appears that different blaCTX-M subgroups have become more prominent durings period.
Carbapenem and colistin are essential treatment options as antibiotics of last resort. It has been observed that Enterobacterales develop resistance to these antibiotics more frequently. ESBL-producing and carbapenem-resistant Enterobacterales can increase colistin use over time despite its nephrotoxic and neurotoxic effects and its narrow spectrum of activity (11). WHO (12) reports carbapenem-resistant and colistin-resistant Enterobacterales as a critical global public health threat. Conversely, Jeon et al. (59) investigated imipenem resistance in Salmonella strains isolated from retail chicken meat using disc diffusion and reported that 1.8% of the strains were resistant. In another study, imipenem and carbapenem resistance were examined, and 0.2% of carbapenem-resistant Salmonella strains were isolated from individuals under 18 (60). Li et al. (61) examined the presence of the mcr-1 gene in Salmonella strains from retail food products and food poisoning cases. While they did not find mcr-1 in 950 Salmonella strains collected between 2006 and 2011, they reported that 3% of 333 Salmonella strains isolated in 2012–2015 contained mcr-1. Casagrande Proietti et al. (62) analyzed colistin resistance in 85 Salmonella Infantis strains, and 3.5% showed phenotypic colistin resistance (MIC test). The mcr-1.1 variant was found in one Salmonella Infantis strain, and the mcr-1.2 variant in two strains, using WGS and PFGE methods. The fact that carbapenem- and polymyxin-group antibiotics are not frequently used in the poultry sector in Türkiye may explain the absence of resistance to these groups.
Foodborne salmonellosis cases pose a threat to public health, and inadequate heat treatment along with cross-contamination in poultry meat and meat products increase this risk. This study identified Salmonella serotypes from chicken meat that can be particularly harmful to human health. For risk assessment, the likelihood of Salmonella-related foodborne illness was considered low because the overall consumption frequency of chicken was very low, regardless of consumption patterns or cooking methods. The study noted that S. Infantis was the dominant serotype isolated from chicken meat in Istanbul. This finding aligns with recent studies conducted in Türkiye and neighboring countries in the Balkan region, Europe. However, a shift in the dominant Salmonella serotype could pose new health risks compared to previous research. Despite growing concerns about foodborne illnesses caused by Salmonella from poultry consumption, many studies in Türkiye suggest that the risk remains relatively acceptable. Additionally, Salmonella serotypes showed resistance to several antibiotics used to treat salmonellosis. They also complicate treatment by spreading antibiotic resistance through mobile genetic elements such as plasmids. The detection of ESBL-producing Salmonella strains presents a serious public health threat. The rates of carbapenem and colistin-resistant Salmonella strains are lower than those observed in E. coli and K. pneumoniae, other members of Enterobacterales, which show higher resistance rates. It is crucial for public health to avoid the unnecessary use of last-resort antibiotics like carbapenems and colistin and to monitor resistance development. Finally, the study found that the rate of MDR S. Infantis was relatively high. Therefore, adopting the ‘One Health’ approach is essential to prevent further public health damage by controlling antibiotic use.
Author’s note
This study is based on the Ph.D. thesis of the first author, Selman Bahadır Orhan.
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, CP193634.1; https://www.ncbi.nlm.nih.gov/genbank/, CP193634.1.
Author contributions
SO: Investigation, Resources, Writing – original draft, Writing – review & editing. AS: Conceptualization, Formal analysis, Investigation, Software, Visualization, Writing – original draft, Writing – review & editing. AA: Conceptualization, Data curation, Formal analysis, Investigation, Project administration, Resources, Software, Supervision, Visualization, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Acknowledgments
The authors would like to acknowledge Zhanylbubu MAMATOVA (Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Istanbul University-Cerrahpasa) and Oğuzhan BAYGUN (Istanbul Medeniyet University) for technical assistance.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Footnotes
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Keywords: chicken meat, extended-spectrum beta-lactamase, multidrug resistance, Salmonella serotypes, sanger sequence
Citation: Orhan SB, Suleymanoglu AA and Aydin A (2026) Genetic diversity with potential ESBL-producing and multidrug-resistant Salmonella strains from chicken meat. Front. Vet. Sci. 12:1709758. doi: 10.3389/fvets.2025.1709758
Edited by:
Mahamat Fayiz Abakar, Institut de Recherche en Élevage Pour le Développement (IRED), ChadReviewed by:
Yanan Wang, Henan Agricultural University, ChinaSuraia Nusrin, East West University, Bangladesh
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*Correspondence: Ali Aydin, YWxpYXlkaW5AaXN0YW5idWwuZWR1LnRy; YWxpYXlkaW5AaXVjLmVkdS50cg==