Addressing antibiotic resistance has become an international focus as it affects humans, animals, and agricultural systems. In this investigation, E. coli isolates representing different pathogroups recovered from seafood samples collected from Mumbai were examined for their susceptibility to antimicrobials. Considering the persistent contamination of coastal waters in this densely populated metropolitan city, we anticipated an increased incidence of antibiotic-resistant E. coli.

The results of antibiotic susceptibility testing indicated the occurrence of E. coli pathogroups resistant to most clinically relevant antibiotics. The result is alarming, as E. coli in general is intrinsically susceptible to nearly all the antimicrobial agents of clinical significance.¹⁵ However, E. coli is known for its receptive capacity to accumulate resistant genes, especially through horizontal gene transfer; this might have played an important role in its evolution with respect to antimicrobial resistance and its rapid spread among pathogroups.^5,16 For the last decades, the number of resistance genes in E. coli has been steadily increasing, which has made E. coli a bacterium with the highest burden of antibiotic resistance.^17,18

Resistance to third-generation cephalosporins was prevalent (97%) among the pathogenic isolates (Table 2, Figure 1). We observed the highest resistance against cefotaxime (97%) and the lowest resistance against ceftriaxone (66.7%) in our E. coli isolates. Singh et al. reported a similar level of resistance in Enterobacterales isolated from seafood in Mumbai, where a majority (>90%) of the tested isolates showed resistance to cefotaxime, cefpodoxime, and ceftazidime, which are third-generation cephalosporins.¹¹ The percentage of resistance shown by the isolates towards cefotaxime (95%) was high and comparable to our results. High cephalosporin resistance in E. coli isolates from frozen shrimp has been reported from Saudi Arabia.¹⁹ However, this study reported high resistance towards first-generation cephalosporins compared to resistance to third-generation cephalosporins observed in our study. Our study also showed high resistance to aminoglycosides, monobactams, carbapenems, quinolones, and fluoroquinolone antibiotics. Ibrahim and Elhadi reported a different susceptibility pattern for penicillin (ampicillin 90.7%, piperacillin 87.1%), quinolones (nalidixic acid 64.2%), sulfonamides (trimethoprim/sulfamethoxazole 50.7%), and tetracycline (41.4%).¹⁹ Contrary to our findings, a study from China reported high resistance of E. coli isolates isolated from fish and shellfish towards chloramphenicol (72.1%) and tetracycline (93.7%).²⁰ E. coli isolated from fish samples in Cameroon, Africa, showed high resistance to trimethoprim-sulfamethoxazole, ampicillin, and ticarcillin compared to other antibiotics.²¹ Notably, different resistance patterns are usually observed for pathogenic and non-pathogenic strains of E. coli owing to the presence of resistant genes on plasmids. In addition to several plasmid-borne antibiotic resistance genes, E. coli possesses the marRAB locus. This chromosomally encoded intrinsic resistance mechanism confers resistance to various antibiotics, including tetracyclines, chloramphenicol, cephalosporins, nalidixic acid, penicillins, rifampin, and fluoroquinolones.²² Overall, the isolates screened in this present study were largely resistant towards beta-lactam antibiotics, and relatively more sensitive to non-beta-lactam antibiotics. Resistance towards beta-lactam antibiotics is common among bacteria, and its emergence is on the rise due to their widespread use.²³

Among 23 EHEC isolates tested, a large proportion of EHEC/STEC isolates were resistant to 7-20 antibiotics with their MAR indices ranging from 0.33-0.95 (Tables 3 and 4). Some of these included the well-known EHEC serogroups O157 and O26 involved in several food-borne outbreaks. EHEC O26 is an important non-O157 serogroup along with O103, O111, and O145 recognized as emerging, virulent non-O157 EHEC capable of causing bloody diarrhoea and haemolytic uraemic syndrome (HUS).²⁴ Other STEC serogroup such as O7, O20, O149 (Table 3) have been reported to be associated with cattle, which are the major reservoirs of STEC strains.^25,26 Three isolates of EHEC O157, isolated from different seafood samples, were resistant to 7, 17, and 18 antibiotics, respectively (Table 3). The isolation of serogroup O157 resistant to only ciprofloxacin from fish was reported by Onmaz et al. in 2020.²⁷ Two EHEC/STEC isolates were resistant to each antibiotic tested except chloramphenicol. Overall, the tested EHEC isolates showed high resistance towards colistin, aminoglycosides, and lactamase inhibitor (Figure 3). A recent study characterizing STEC isolated from shellfish in Egypt reported resistance to multiple antibiotics, including β-lactams and β-lactam inhibitors, ciprofloxacin, colistin, tetracycline, and fosfomycin.²⁸

Surprisingly, in our study, two EPEC isolates exhibited markedly different resistance profiles. The isolate TIM651 was resistant to 13 antibiotics, while PMH14 was resistant to only three antibiotics (Table 4). Even though many EPEC isolates share similar antibiotic resistance profiles, variations can be expected, as these pathogenic strains are highly diverse in nature.²⁹ Both the isolates showed resistance towards third-generation cephalosporins, monobactam, and colistin (Figure 3). Studies from India suggest that EPEC clinical strains have gained resistance to multiple antibiotics commonly employed in the treatment of diarrheal diseases.^30,31 A study reported total resistance to cephalothin, cefuroxime, and sulfamethoxazole, as well as very high resistance to tetracycline (76.3%) and streptomycin (84.2%) in clinical EPEC isolates.²⁹ In our isolates, tetracycline resistance was not found, and also, only one isolate showed resistance to cephalothin.

Varying antibiotic resistance patterns were observed among the ETEC isolates. The isolate TIE83 with a MAR index of 0.81 was sensitive to cefpodoxime, chloramphenicol, ertapenem, and meropenem, and was resistant to all other 17 antibiotics, including third-generation cephalosporins (Table 4 and Figure 3). In contrast, the isolate TEC19 was resistant to five antibiotics, including cefotaxime, cefoxitin, cefpodoxime, meropenem, and amikacin. Two isolates were resistant to ciprofloxacin in addition to cephalosporins and carbapenems. Since the emergence of ciprofloxacin-resistant ETEC in 2001, there has been a trend of increasing resistance patterns of ETEC to fluoroquinolones.^32,33 High prevalence of resistance to trimethoprim-sulfamethoxazole, ampicillin, and tetracycline was seen among the ETEC isolates obtained from ready-to-eat foods in China.²⁰ This study reports tetracycline resistance as 66.7%, whereas we found 40% resistance against tetracycline. Among 33 isolates screened, 32 isolates showed multidrug-resistance. Studies on the prevalence of seafood-originated drug-resistant E. coli from samples collected from Southern India reported the occurrence of strains with multiple drug resistance, implicating seafood as the carrier of MDR bacteria.^34,35 According to a new investigation on the occurrence of ESBL-producing bacteria in seafood, 169 (78.60%) isolates of different Enterobacterales species showed an ESBL-positive phenotype, with E. coli representing the major species.³⁶ Various ESBL-encoding genes were also identified in these isolates. Further, the occurrence of bla_NDM-harboring E. coli has also been reported in seafood.^10,36 A few other studies have reported the prevalence of antibiotic-resistant E. coli in commercial seafood samples in Korea,^37,38 commercial fish captured from Conception Bay, Chile,³⁹ shellfish from retail markets of Vietnam,⁴⁰ shrimps and shrimp farm environments in Thailand,⁴¹ in oysters and mussels in Atlantic Canada,⁴² and fish from retail markets of Cambodia.⁴³ A study from Mizoram, Northeast India, reported high prevalence of multidrug-resistant E. coli isolates belonging to EPEC and EIEC pathotypes associated with paediatric diarrhoea.³⁰ The MDR phenotype was observed in 41.4% of the isolates, which showed high resistance against cephalosporin drugs, aminoglycosides, carbapenem, fluoroquinolone, and sulphonamides. Multidrug-resistance involving β-lactams, third-generation cephalosporins, piperacillin, levofloxacin, and gentamicin has been described in E. coli pathogroups isolated from diarrheic children in Bihar, India.⁴⁴ Recently, Ghosh et al. reported a high incidence of diarrheagenic E. coli resistant to a minimum of six different classes of antimicrobials.⁴⁵ The endemicity of different pathogroups of E. coli means that they could be found in the environment and consequently in foods, including seafood, when the sanitation infrastructure is inadequately disproportional to the population, particularly in developing nations.⁴⁶ All these studies highlight the exposure of seafood to highly antibiotic-resistant E. coli from diverse sources, including humans and animals, and the need to identify contaminated sources and contain the spread of MDR pathogens via seafood.

An interesting observation from this study was the increased sensitivity to antibiotics such as chloramphenicol, co-trimoxazole, and tetracycline (Figure 1). The clinical application of these antibiotics has declined significantly over the last three decades due to the development of widespread bacterial resistance.^47-49 The increased susceptibility of diarrheagenic E. coli observed in this study warrants further investigation to understand the factors that have contributed to the susceptibility of E. coli to these antibiotics, particularly in light of the drastic rise in resistance to other antibiotics, such as β-lactams, aminoglycosides, and fluoroquinolones.

The MAR indices of 33 isolates ranged from 0.09 to 0.95 (Table 3), suggesting that these strains were from a high-risk environment where they were exposed to higher levels of antibiotics due to extensive use. A similar range of MAR index, extending to 1.0 from 0.09, was also reported from the same location in seafood samples.¹¹ The antibiotic resistance patterns of seafood isolates of this study are comparable with clinical isolates of pathogenic E. coli. The multidrug-resistance traits reported in clinical isolates of diarrheagenic E. coli in India suggest that these strains have a human reservoir, and enter the aquatic environment through various routes of contamination.