Abstract
Foodborne pathogens' transmission is essential in the spread of antibiotic resistance, and extended-spectrum beta-lactamase-producing Escherichia coli especially threatens public health. E. coli plays an essential role in the resistance to commonly used beta-lactam group antibiotics. Ready-to-eat (RTE) stuffed mussels are among many restaurants and street vendors, presenting potential health risks of food hygiene origin. 200 RTE stuffed mussels were collected from the Asian and European sides of Istanbul and analysed for the presence of E. coli. As a result of PCR analysis, E. coli was detected in 7 (3.5%) samples. An antibiotic susceptibility test was performed using the disc diffusion method to determine ESBL and carbapenem resistance. All isolates were resistant to ampicillin. The double-disk synergy test was performed as an ESBL phenotypic confirmation test, and no phenotypically ESBL-producing E. coli were detected. The blaTEM gene was detected in one isolate (14.2%) by mPCR, but blaCTX-M, blaSHV, and blaOXA genes were not observed. Meropenem and imipenem were used with the disk diffusion method for carbapenem resistance study, and no resistant isolate was found. Carbapenem resistance genes were investigated by monoplex PCR, and blaNDM-1, blaOXA-48, blaVIM, and blaIMP resistance genes were not detected. This is the first report on ESBL-producing E. coli in RTE stuffed mussels in Türkiye, which draws attention to a public health risk.
1 Introduction
Antibiotic resistance has become a global health threat, and its importance is increasing. Food-borne mechanisms are also involved in the spread of this health concern. Various bacteria have great potential in this regard. People can be exposed to multiple antibiotic-resistant bacteria through food (Tiedje et al., 2023).
Escherichia coli (E. coli) is a Gram-negative bacterium with few pathogenic types, most of which are commensal. Pathotypes of E. coli found naturally in the intestinal flora can cause serious infections. E. coli spread is likely found in food contaminated with faeces (Kaper et al., 2004). WHO (2017) classified Enterobacterales resistant to third-generation cephalosporins and carbapenems as critical priority pathogens. E. coli poses a significant risk to global health due to its prevalence, multidrug resistance, and rapid spread of antimicrobial resistance to other organisms (Bush, 2010).
Antibiotic resistance can be spread through various genetic materials. With the progress in genetic analysis methods, many antibiotic-resistance genes have been detected (Allen et al., 2010), and the spread of extended-spectrum beta-lactamases (ESBLs) through genetic material is a concern for public health. E. coli has attracted attention with horizontal gene transfer and is one of the leading bacteria behind the increase of multiple antibiotic resistance.
Carbapenems are antibiotics used in the treatment of ESBL-producing bacteria. Therefore, resistance to carbapenems is very concerning. Carbapenem resistance is an increasing health concern, and treatment against infections caused by bacteria with this resistance is limited today (Süleymanoğlu et al., 2022). WHO (2017) states that carbapenem-resistant bacteria are a significant threat to global health, and classifies them as the pathogens with the most critical priority. In many countries, carbapenem group antibiotics are not permitted for use in livestock.
Humans, animals, and the environment interact in the world. Antimicrobial resistance can be transferred during this interaction. Therefore, the concept of One-Health is one of the popular subjects in public health. The One-Health phenomenon aims to reduce the amount of antimicrobial resistance (AMR) and reduce deaths due to it. With One-Health approach, it is possible to prevent or reduce the rate of increasing antibiotic resistance and bacteria that can spread rapidly through foods (Aydin and Sudagidan, 2022).
This study aimed to determine the presence of E. coli in ready-to-eat stuffed mussels in Istanbul. In addition, it analysed whether the isolates produced ESBL and whether they were resistant to carbapenems. The aim was to investigate the possibility that ready-to-consume end products harbor antibiotic-resistance genes that can be carried through food. The presence of E. coli also sheds light on the level of contamination in foods.
2 Materials and methods
2.1 Sampling
200 ready-to-eat stuffed mussel samples were delivered to the laboratory, and all samples were aseptically packed in sterile sampling bags from 100 districts located on the coastal strip of the Marmara Sea on the Asian and European sides of Istanbul province. Samples were collected from street vendors (n = 35), kiosks (n = 35), and restaurants (n = 30) for each side from June to October 2022. All samples were transported in thermal boxes (≤+4 °C) to the laboratory and were processed immediately upon arrival.
2.2 Isolation and identification of E. coli
Reference cultures: E. coli ATCC 25922, Klebsiella pneumoniae NCTC 13443 with NDM-1 metallo beta lactamase.
RTE stuffed mussel samples were delivered to the laboratory through cold chain under aseptic conditions. The isolation and identification of E. coli was performed according to ISO 16649-1 standard method (ISO, 2018). 10 g sample was weighed, 90 mL Buffered Peptone Water (Oxoid CM 0509, UK) was added, and the suspension was mixed in a stomacher (Interscience, France). The serial decimal dilutions were plated on Tryptone Bile Glucuronide (TBX; Oxoid, CM 0945) agar and incubated at 41 ± 1 °C for 18–24 h. Suspected E. coli colonies (blue-green with an opaque halo) were transferred onto Eosin Methylene Blue (EMB; Merck, 103,858) agar and incubated at 37 °C for 1–24 h. After the isolation, E. coli strains were plated for purity testing, a single colony was streaked onto Tryptic Soy Agar (TSA; Oxoid, CM 0131) and incubated at 37 °C for 18–24 h.
2.3 Verification of E. coli isolates by PCR
For molecular genetic verification, bacterial DNA extraction was performed (Liu et al., 2004). PCR targeting the E. coli specific primers ECO-1 and -2 (ECO-1; 5′-GACCTCGGTTTAGTTCACAGA-3′ and ECO-2; 5′-CACACGCTGACGCTGACCA-3′ (585 bp)) was performed to confirm the suspected E. coli isolates (Schippa et al., 2010). The cycle condition for ECO-1 and -2 primers: initial denaturation of 1 cycle at 95 °C for 5 min; annealing 30 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 45 s; extension 72 °C for 7 min and 10 °C for infinity in a thermal cycler (Applied Biosystems, Veriti, USA). Amplification products were analysed in 1.5% (w/v) agarose gel containing 5 µL SafeView DNA stain (ABM, Canada) in the Infinity Gel Imaging System (Vilber Lourmat, France).
2.4 Antibiotic susceptibility test
The disc diffusion method was chosen for antibiotic susceptibility testing on Mueller–Hinton Agar (MHA; Oxoid, CM 337). For this purpose, cefotaxime (CTX) (Oxoid, CT0166B, 30 µg), ceftazidime (CAZ) (Oxoid, CTO412B, 30 µg), amoxicillin-clavulanic acid (AMC; Oxoid, CT0223B, 30 µg), meropenem (MEM) (Oxoid, CT0774B, 10 µg), and ampicillin (AMP; Oxoid, CTOOO3B, 10 µg) antibiotic discs were used. The plates were incubated at 35 ± 2 °C for 18 ± 2 h. The results were evaluated according to EUCAST (2022) and CLSI (2021) clinical breakpoint guidelines.
2.5 Phenotypic ESBL confirmatory test
The double disc synergy test (DDST) was used for ESBL phenotypic confirmation testing. CAZ, CTX, and AMC antibiotic discs were positioned at distances of 20 mm on MHA, the AMC disc being in the middle. The plates were incubated at 35 ± 2 °C for 24 h. For this purpose, the test was performed according to EUCAST guidelines (2017).
2.6 Detection of ESBL genes
ESBL genes were detected in E. coli strains by mPCR (Table 1). The cycle conditions for multiplex PCR of blaSHV, blaTEM, blaCTX-M, and blaOXA (Table 1) were initial denaturation 1 cycle at 95 °C for 5 min; annealing 35 cycles of 95 °C for 30 s, 51 °C for 30 s, 72 °C for 45 s; extension 72 °C for 7 min, and 10 °C for infinity in a thermal cycler. Amplification products were analysed in 1.5% (w/v) agarose gel containing 5 µL SafeView DNA stain (Fang et al., 2007).
Investigated ESBL genes
| Target gene | Primer sequence | Melting temperature Tm (°C) | Product size (bp) |
| blaSHV | SHV_F 5′-CTTTATCGGCCCTCACTCAA-3′ SHV_R 5′-AGGTGCTCATCATGGGAAAG-3 | 51 | 237 |
| blaTEM | TEM_F 5′-CGCCGCATACACTATTCTCAGAATGA-3′ TEM_R 5′- ACGCTCACCGGCTCCAGATTTAT-3′ | 51 | 445 |
| blaCTX-M | CTX-M_F 5′-ATGTGCAGYACCAGTAARGTKATGGC-3′ CTX-M_R 5′-TGGGTRAARTARGTSACCAGAAYCAGCGG-3′ | 51 | 593 |
| blaOXA | OXA_F 5′-ACACAATACATATCAACTTCGC-3′ OXA_R 5′-AGTGTGTTTAGAATGGTGATC-3′ | 51 | 813 |
2.7 Detection of carbapenemase resistance genes
For the detection of this carbapenemase gene, monoplex PCR was used. The cycle condition for blaNDM-1, blaOXA-48, blaVIM, and blaKPC was conducted as an ECDC (2019) manual for detecting carbapenem resistance (Table 2). Initial denaturation 1 cycle at 94 °C for 5 min; annealing 30 cycles of 94 °C for 30 s, 52 °C for blaVIM; 58 °C for blaOXA-48 and blaNDM; 60 °C blaKPC for 30 s, 72 °C for 60 s; extension 72 °C for 10 min, and 10 °C for infinity in a thermal cycler. Amplification products were analysed in 1.5% (w/v) agarose gel containing 5 µL SafeView DNA stains.
Investigated carbapenem resistance genes
| Target gene | Primer sequence | Melting temperature Tm (°C) | Product size (bp) |
| blaOXA-48 | OXA_F 5′-TTGGTGGCATCGATTATCGG-3′ OXA_R 5′-GAGCACTTCTTTTGTGATGGC-3′ | 58 | 744 |
| blaNDM | NDM_F 5′-TGGCAGCACACTTCCTATC-3′ NDM_R 5′-AGATTGCCGAGCGACTTG-3′ | 58 | 488 |
| blaKPC | KPC_F 5′-CTGTCTTGTCTCTCATGGCC-3′ KPC_R 5′-CCTCGCTGTRCTTGTCATCC-3′ | 60 | 796 |
| blaVIM | VIM_F 5′-AGTGGTGAGTATCCGACAG-3′ VIM_R 5′-TCAATCTCCGCGAGAAG-3′ | 52 | 212 |
3 Results and discussion
The World Health Organisation emphasises Enterobacterales, especially E. coli and K. pneumonia carbapenem-resistant and third-generation cephalosporin-resistant isolates, as antibiotic-resistant bacteria that should be monitored as a priority (WHO, 2017). Various antibiotic-resistant bacteria in food may infect consumers due to poor sanitation and cross-contamination (FAO/WHO, 2019). In developing countries, aquaculture is often done under unhealthy conditions.
9 (9/200, 4.5%) suspected E. coli isolates were detected from RTE stuffed mussels using conventional methods. PCR was used to confirm the suspected E. coli isolates. An E. coli-specific 16S rRNA gene was detected in 7 (7/200, 3.5%) isolates. 3 (3/100, 3%) E. coli isolates were obtained from the European side samples. All E. coli isolates from the European side were obtained from street vendors and kiosks in the same district. In addition, 4 (4/100, 4%) E. coli isolates were obtained from the Asian side. Only 2 E. coli isolates from the Asian side were obtained from street vendors, and 2 isolates originated from kiosks. According to the sales points, 4 E. coli isolates were obtained from street vendors (4/70, 5.7%) and 3 (3/70, 4.2%) from kiosks. In addition, no E. coli isolates were isolated from restaurants (0/60, 0%). Many researchers found no critical foodborne pathogens in their investigated seafood (Morrison et al., 2015; Aydin et al., 2023). Since the E. coli isolates examined in this study were not serotyped, whether they were pathogenic is unknown. However, in RTE products, E. coli, an indicator of fecal contamination, threatens public health. The present study differs with the detection of 3.5% E. coli in RTE stuffed mussels. The fact that the E. coli isolates in this study were obtained from kiosks and street vendors, not restaurants, strengthens the possibility of E. coli contamination due to poor hygiene conditions.
All isolates (100%) were susceptible to MEM, CTX, and CAZ. In contrast to their susceptibility to other antibiotics, all isolates were resistant to AMP (100%), and 85.7% were resistant to AMC according to EUCAST (2022). However, none of the isolates phenotypically produced extended-spectrum beta-lactamase by DDST. Ampicillin resistance was detected in all E. coli isolates in this study. Although there is a high correlation between ampicillin resistance and the blaTEM gene in general, this correlation was found to be low in this study. Indeed, in a study conducted in Spain, the blaTEM gene was reported in 103 (83%) of 124 ampicillin-resistant E. coli (Briñas et al., 2002). However, the lack of studies on E. coli isolates obtained from mussels makes it challenging to explain this difference. On the other hand, the results of our study are similar to Briñas et al. (2002) regarding the presence of blaOXA and blaSHV genes. The fact that all isolates in this study were resistant to ampicillin, one of the beta-lactam group antibiotics, once again reveals the threat posed by ESBL-producing E. coli to public health, as stated by WHO.
Currently, ESBL-producing E. coli studies in ready-to-eat stuffed mussels are limited. Le et al. (2015) isolated 18.3% (11/60) ESBL-producing E. coli from shrimps. In another study, Van et al. (2008) found the blaTEM gene in 5 (10%) E. coli isolated as a result of their analyses on 50 shellfish and reported that 5 (100%) isolates were resistant to AMP and 3 (60%) isolates were resistant to AMC. This study's results were similar to those of Le et al. (2015) and Van et al. (2008). Moreover, it is seen that resistance to the penicillin group is common, especially in shellfish. Resistance to beta-lactamase inhibitors such as clavulanic acid is a phenomenon of global health concern.
The carbapenem group is commonly used in the treatment of ESBL-producing Enterobacterales. The fact that few antibiotics are available against ESBL-producing bacteria makes carbapenemases a critical issue for public health (Süleymanoğlu et al., 2022; Aydin et al., 2024). In a study, Roschanski et al. (2017) found that one E. coli isolate carried the blaVIM gene in 45 Enterobacterales examined in retail seafood. Remarkably, this strain was not resistant to imipenem and meropenem determined according to CLSI guidelines with the disc diffusion method. In another study, Morrison et al. (2015) isolated 4 (3.3%) carbapenem-resistant bacteria in 121 seafood products. The isolated bacteria were determined as Stenotrophomonas, Myroides, and Pseudomonas spp.; all strains contained blaOXA-48. Moreover, Aydin et al. (2023) found 28.3% resistance to doripenem in Pseudomonas spp. (Gram-negative bacteria like E. coli) due to metagenomic analyses in sea bass using the disc diffusion method. These results indicate seafood's resistance to the carbapenem group.
Celik et al. (2023) collected 96 raw shrimp and 96 raw mussel samples from the Marmara Sea coastline (n = 21) and fish markets (n = 75) in Istanbul in 2017 and isolated 17.7% E. coli isolates (34/192). However, in the present study, stuffed mussels were collected from restaurants, street vendors, and kiosks and 3.5% E. coli were determined. Celik et al. (2023) analysed raw shrimp and mussel samples and sampling points using biochemical tests such as Gram staining, IMVIC, oxidase test, and carbohydrate fermentation test to confirm E. coli isolates. In our study, suspected E. coli strains were verified with E. coli-specific 16S rRNA by PCR. The difference in ESBL genes between the two studies can be explained by the sampling date, sample collection area, sampling points (markets, etc.), and the examination method of ready-to-eat mussels. However, the similar rate of blaTEM gene in both studies draws attention to the risk of contamination and the extent of public health threat posed by the final products. It is a promising finding in terms of public health that carbapenem-resistance genes were not reported. However, the similarity of the reported blaTEM rate indicates that the future presence of carbapenem-resistance genes is an essential threat to public health. An isolate obtained from an Asian street vendor contained the blaTEM gene. None of the E. coli isolates in this study contained blaCTX-M, blaOXA, blaNDM, blaSHV, blaVIM, or blaKPC genes. Furthermore, the blaTEM gene isolate (Sampling no: 174) was resistant to AMP and AMC (Table 3).
Origins and antibiotic resistance properties of E. coli isolates from ready-to-eat stuffed mussel
| Sample ID | Side of Istanbul | Source of sample | Antibiotic resistance | ESBL genes |
| S93 | Europe | Peddlers | AMP, AMC30* | – |
| S94 | Europe | Kiosk | AMP, AMC30* | – |
| S95 | Europe | Kiosk | AMP, AMC30* | – |
| S127 | Asia | Peddlers | AMP | – |
| S132 | Asia | Peddlers | AMP, AMC30* | – |
| S141 | Asia | Peddlers | AMP, AMC30* | – |
| S174 | Asia | Kiosk | AMP, AMC30* | blaTEM |
| S199 | Asia | Peddlers | AMP, AMC30* | – |
*It is resistant according to EUCAST (2022) guidelines but not to CLSI (2021).
4 Conclusions
Plasmids make a significant contribution to the spread of ESBL and carbapenem resistance. In this study, for the first time in Türkiye, the presence of the blaTEM gene was reported in RTE mussels, indicating the prevalence of resistance. The faecal contamination indicator E. coli in RTE stuffed mussels indicates that general hygiene rules are not followed when selling this food product. In this regard, further studies are needed to better understand the spread of antibiotic resistance in sea products. However, the collaboration of veterinarians and medical doctors on this issue will slow the spread of antibiotic resistance. Therefore, the One Health topic should be considered, and the last weapons of health science, such as carbapenems, should be protected.
Ethical statement
The study does not require ethical approval from any ethics committee.
Author contributions
Conceptualization, A.A., A.A.S., and S.Ö.; writing, A.A., and A.A.S.; investigation, S.Ö., and A.A.S.; resources, S.Ö., and A.A.S.; original draft preparation, A.A. and A.A.S; writing — review and editing, A.A., and A.A.S.; supervision, A.A.; project administration, A.A. All authors have read and agreed to the published version of the manuscript.
Data availability statement
The data presented in this study are available on request from the corresponding authors.
Funding sources
This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK 2209-A) to support Sezer OZKAN in a University Student Research Project Support Program.
Conflicts of interest
The authors declare that there is no conflict of interest.
Acknowledgment
The authors would like to acknowledge Zhanylbubu Mamatova (Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Istanbul University-Cerrahpasa) for her technical assistance.
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