High Prevalence of Multiple Antibiotic-Resistant, Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli in Fresh Seafood Sold in Retail Markets of Mumbai, India

In this study, fresh seafood in retail markets was investigated for the antibiotic susceptibility patterns of the faecal indicator Escherichia coli and distribution of important β-lactamase encoding genes. E. coli were isolated from 50 (37 fish and 13 shellfish) fresh seafood samples and studied with respect to the phenotypic and genotypic characters of antibiotic resistance. Of 475 E. coli isolates from fresh seafood, 71.58% exhibited extended-spectrum β-lactamase (ESBL)-positive phenotypes. A high percentage of isolates were resistant to indicator cephalosporins cefotaxime (95%), cefpodoxime (90.88%) and ceftazidime (90.29%). Relatively higher susceptibilities were recorded against imipenem (74.41%), cefoxitin (66.76%) and meropenem (51.18%). The multiple antibiotic resistance (MAR) index of 97.35% of the isolates was above 0.18. The ESBL genes blaCTX-M, blaSHV and blaTEM were detected in 62.37%, 23.35% and 2.6% of E. coli isolates, respectively. The ESBL-producing isolates also harboured the metallo-β-lactamase-encoding genes blaOXA (7.06%), blaNDM (4.42%) and blaVIM (0.88%). This study highlights the risk of dissemination of multidrug resistant E. coli in seafood consumer communities and also the need to improve the hygiene of the coastal waters, landing centres and the retail markets.


Introduction
Escherichia coli is a common inhabitant of the gastrointestinal tract of both humans and animals. Although E. coli is popular as an indicator of faecal contamination of water and foods, it is a significant food-borne human pathogen [1]. Contamination of seafood with E. coli as a consequence of contamination of coastal waters with domestic sewage is common in densely populated countries like India [2,3]. Such pathogenic E. coli can cause a range of infections, such as gastroenteritis, wound infections, septicaemia, urinary tract infections, etc. [4].
Resistance to multiple, clinically relevant antibiotics in E. coli has severely compromised the efficacy of antibiotic chemotherapy. Multidrug-resistant (MDR) E. coli is not restricted to clinical settings alone. Since humans and animals are sources of contamination of aquatic environments, MDR E. coli can be often found in seafood. Seafood as a vehicle of MDR E. coli is a serious concern, since such bacteria can be introduced via seafood to distant geographical regions, where such resistance patterns were unknown before [5]. Globally, beta-lactam antibiotics are among the most commonly prescribed drugs, which include penicillins, cephalosporins, cephamycins and carbapenems. In E. coli, as well as in several other members of the Enterobacteriaceae, production of one or more types of β-lactamases that hydrolyse β-lactam antibiotics is a key mechanism of resistance [6,7]. Among these,

Sample Collection, Isolation and Identification of Escherichia coli From Seafood
Fresh seafood samples (n = 50) comprising of fish (37) and shrimps (13) were collected from retail markets in north-western Mumbai, India, during October 2016 to February 2018. The samples were transported immediately to the laboratory in a chilled condition in sterile containers and processed for the isolation of E. coli. Equal portions from the head, belly and tail regions of finfish were pooled and 25 g of this pooled sample was used for analysis. Clams were opened using a sterile scalpel and the meat was collected from 2-3 specimens and pooled. Similarly, in the case of shrimps, the meat was collected and pooled. Twenty-five grams of the pooled meat was homogenized in 225 mL of tryptone broth (Hi-Media, Mumbai, India) in a stomacher (Seward Stomacher 80, Lab system, London, UK), serially diluted in tryptone water and spread plated on MacConkey agar (Hi-Media, Mumbai, India). Typical pink colonies, 3-5 from the useful dilution, were purified on Luria Bertani (LB) agar. Oxidase-negative isolates were subjected to biochemical tests for the identification of E. coli [23].

Detection of ESBL and MBL Phenotypes
For phenotypic detection of ESBL production, the double disc synergy test was followed [26]. The test bacterium was grown in Mueller-Hinton (MH) broth to 0.5 McFarland units and a 0.1 mL of the broth culture was spread plated on MH agar. An amoxicillin/clavulanic acid (30/10 µg) disc was placed at the centre of the plate and ceftazidime (30 µg), cefotaxime (30 µg) and cefpodoxime (10 µg) discs were placed 20-30 mm away from the central disc. An extension in the zone of inhibition around the peripheral disc towards the centrally placed amoxicillin/clavulanic acid disc by at least 5 mm was considered as due to ESBL production. For further confirmation of an ESBL + phenotype, the Triple ESBL detection Ezy MIC TM Strip (Hi-Media, Mumbai, India) was used according to the manufacturer's instructions. The upper half of the strip designated as "mixture + " is coated with ceftazidime, cefotaxime and cefepime plus clavulanic acid, while the lower half of the strip designated as "mixture" is coated with the same antibiotics in a reverse concentration gradient, but without an inhibitor. The strip was placed on the lawn culture of each test strain on MH agar and minimum inhibitory concentrations (MIC)s were read from the mixture and mixture + ends of the strip. The isolate was considered ESBL-positive if the mix/mix + value was ≥8. AmpC β-lactamase was inhibited by adding 200 µg/mL cloxacillin in the agar medium.
For detection of metallo-β-lactamase (MBL) production, the double disc diffusion synergy test was used [27]. The test bacterium was grown in Muller-Hinton (MH) broth as described above and spread onto MH agar plates. Two imipenem discs (10 µg), one of which contained 10 µL of 0.1 M EDTA (292 µg), were placed 25 mm apart on the inoculated MH plates. MBL production was defined by a zone of inhibition of ≥4 mm around the imipenem-EDTA disc compared to the disc containing imipenem alone.

Results and Discussion
Antibiotic-resistant bacteria are frequently encountered in raw seafood in India [20,[34][35][36] and the incidence is alarmingly high in seafood harvested off the Mumbai coast [22,37]. The coastal city of Mumbai is densely populated, and the numerous creeks that lead to the sea act as conduits for draining untreated sewage. Every day, an estimated 2000 million litres of sewage is released into the Arabian sea. Direct contamination occurs from the population that live on the beaches without proper sanitation facilities. Retail markets too lack basic facilities to prevent contamination of fish and maintain hygiene. As a result, contamination of seafood with pathogenic and antibiotic-resistant bacteria is expected to occur right from the harvesting stage, till it reaches the consumer.
In this study, we investigated the occurrence of antibiotic-resistant E. coli in seafood, which directly represents the human cause of seafood contamination. The details of 50 seafood samples (

Antibiotic Resistance Profiles of E. coli
The antibiotic resistance patterns of 340 ESBL + isolates are shown in Table 2. Studies reporting isolation of ESBL-producing E. coli from seafood in India are rare. We recently reported that 78.6% of the enterobacteria isolates from fresh seafood in Mumbai, India, were ESBL-positive [22]. ESBL + Gram-negative bacteria were also isolated from effluents of a fish processing plant in Mangalore, India [38]. The latter study suggests that the contaminated fish can again be a source of environmental contamination with antibiotic-resistant bacteria, highlighting the need to treat effluents from fish processing plants. In Germany, ESBL-and AmpC-producing Enterobacteriaceae were detected in 21.3% of seafood samples, with Klebsiella pneumoniae and E. coli being the predominant species [39]. Sellera and colleagues [40] reported isolation of ESBL (CTX-M)-producing E. coli from wild fishes of a polluted area in the South Atlantic coast of Brazil. ESBL-producing E. coli of identical genotypes have been reported from fish, chicken and infected patients in Cambodia [41].  Figure 1A). The isolates were largely susceptible to carbapenems and ciprofloxacin. The bla SHV gene was detected in 79 (23.35%) isolates, 64 of which were from finfish and 15 from shellfish ( Table 3). The isolates were commonly resistant to cefotaxime and ceftazidime ( Figure 1B). Fifty-four (44%) isolates were susceptible to cefoxitin ( Figure 1B). The bla TEM gene was detected in the least number of isolates (9, 2.64%) ( Table 3). The antibiotic susceptibility patterns of bla TEM -positive E. coli are shown in Figure 1C.   The CTX-M group of enzymes, which are commonly found in E. coli, confer resistance to cefotaxime, ceftriaxone and aztreonam [12,15]. Although CTX-M was originally found to be less active against ceftazidime, its variants, such CTX-M-15, hydrolyse ceftazidime efficiently [42][43][44]. In our study, 141 of 212 (66%) bla CTX-M -positive E. coli were susceptible to cefoxitin ( Figure 1A). ESBL-producing E. coli is generally susceptible to cephamycins, thus making cephamycins, such as cefoxitin, viable options for treating ESBL-producing E. coli infections [45]. However, cefoxitin resistance due to loss of a porin has been reported in K. pneumonia and E. coli [46]. TEM, SHV and CTX-M-producing organisms have been reported from a variety of food-producing animals, such as poultry, swine, bovine, horse, rabbit, ostrich, wild boars, etc. [47].
In the present study, the bla CTX-M -harbouring E. coli were predominantly isolated compared to the bla SHV -or bla TEM -harbouring E. coli. The CTX-M group has the highest number of variants, with E. coli being the major producer of this enzyme [48]. The extraordinary mobility of plasmid and/transposon-located bla CTX-M genes within clones of E. coli that are successful as pathogens has made CTX-M β-lactamase the most dominant ESBL currently [48]. The high prevalence of E. coli producing CTX-M β-lactamase in seafood might reflect on the local antibiotic consumption patterns as well as specific resistant clones circulating in the community.

Detection of Carbapenemase-Encoding Genes
Carbapenem-resistant Enterobacteriaceae (CRE) have become the biggest challenge in healthcare settings, as these bacteria are recalcitrant to most of the common antibiotics used. In our study, 21 (4.42%) isolates, 17 from fish and 4 from shellfish, harboured the bla NDM gene ( Table 3). The isolates were resistant to all tested antibiotics except colistin ( Figure 2B). The present study strengthens our previous report of an alarmingly high occurrence of bla NDM -harbouring E. coli in fresh seafood [21,22]. poultry, swine, bovine, horse, rabbit, ostrich, wild boars, etc. [47].
In the present study, the blaCTX-M-harbouring E. coli were predominantly isolated compared to the blaSHVor blaTEM-harbouring E. coli. The CTX-M group has the highest number of variants, with E. coli being the major producer of this enzyme [48]. The extraordinary mobility of plasmid and/transposon-located blaCTX-M genes within clones of E. coli that are successful as pathogens has made CTX-M β-lactamase the most dominant ESBL currently [48]. The high prevalence of E. coli producing CTX-M β-lactamase in seafood might reflect on the local antibiotic consumption patterns as well as specific resistant clones circulating in the community.

Detection of Carbapenemase-Encoding Genes
Carbapenem-resistant Enterobacteriaceae (CRE) have become the biggest challenge in healthcare settings, as these bacteria are recalcitrant to most of the common antibiotics used. In our study, 21 (4.42%) isolates, 17 from fish and 4 from shellfish, harboured the blaNDM gene ( Table 3). The isolates were resistant to all tested antibiotics except colistin ( Figure 2B). The present study strengthens our previous report of an alarmingly high occurrence of blaNDM-harbouring E. coli in fresh seafood [21,22].
The blaOXA and blaVIM genes were detected in 7.06% and 0.88% of the isolates, respectively. The antibiotic resistance profiles of these isolates are shown Figure 2. The blaVIM was first reported in Pseudomonas aeruginosa [49]. Of the three isolates harbouring blaVIM, two isolates were resistant to all 11 antibiotics, while one had intermediate resistant to ceftazidime ( Figure 2C). The VIM type metallo-β-lactamases are mostly reported from Pseudomonas aeruginosa, and from several non-related bacterial species like Achromobacter xylosoxidans, E. coli, Klebsiella pneumoniae, etc., which suggests its horizontal transfer ability among non-related species [50]. Worldwide, blaVIM-2 has been reported to be the dominant MBL variant [51]. Studies from India have reported VIM lactamase producing clinical isolates of P. aeruginoasa [52], Acinetobacter baumannii [53] and E. coli [54]. We did not find any reports of food-associated Gram-negative bacteria harbouring VIM and OXA type β-lactamase genes from India. Elsewhere, a recent study has reported the presence of blaVIM in a food-associated Vibrio parahaemolyticus [55]. In our study, we screened E. coli isolates for ESBL production first using indicator cephalosporins, and this might have resulted in underestimation of certain ceftazidime-susceptible carbapenemase-producing E. coli (e.g., OXA-48) that lack ESBL and AmpC enzymes [56]. The bla OXA and bla VIM genes were detected in 7.06% and 0.88% of the isolates, respectively. The antibiotic resistance profiles of these isolates are shown Figure 2. The bla VIM was first reported in Pseudomonas aeruginosa [49]. Of the three isolates harbouring bla VIM , two isolates were resistant to all 11 antibiotics, while one had intermediate resistant to ceftazidime ( Figure 2C). The VIM type metallo-β-lactamases are mostly reported from Pseudomonas aeruginosa, and from several non-related bacterial species like Achromobacter xylosoxidans, E. coli, Klebsiella pneumoniae, etc., which suggests its horizontal transfer ability among non-related species [50]. Worldwide, bla VIM-2 has been reported to be the dominant MBL variant [51]. Studies from India have reported VIM lactamase producing clinical isolates of P. aeruginoasa [52], Acinetobacter baumannii [53] and E. coli [54]. We did not find any reports of food-associated Gram-negative bacteria harbouring VIM and OXA type β-lactamase genes from India. Elsewhere, a recent study has reported the presence of bla VIM in a food-associated Vibrio parahaemolyticus [55]. In our study, we screened E. coli isolates for ESBL production first using indicator Vet. Sci. 2020, 7, 46 8 of 13 cephalosporins, and this might have resulted in underestimation of certain ceftazidime-susceptible carbapenemase-producing E. coli (e.g., OXA-48) that lack ESBL and AmpC enzymes [56].

Multidrug Resistance in Seafood E. coli
Of the 340 isolates, 331 (97.35%) were resistant to two or more antibiotics with a MAR index of >0.18 (Table 4). A MAR index of more than 0.2 suggests contamination from high-risk sources where antibiotics are extensively used [25], and the majority of our isolates belonged to this category. All (100%) of the E. coli isolates recovered from nine samples were multidrug-resistant. These samples consisted of fish (Sardinella longiceps, Otolithes cuvieri, Epinephelus diacanthus, Mene maculata, Priacanthus hamrur and Pampus argenteus) and shellfish, such as crustaceans (Acetes indicus and Metapenaeus dobsoni) and clams (Meretrix meretrix). Twenty-one E. coli isolates were resistant to the entire range of 11 antibiotics tested. 1.00 21 (6.18) In this study, we analysed 50 samples of fish and shellfish that belonged to 17 different species. Since we isolated multiple isolates of E. coli from a single sample, we selected one E. coli isolate from each species of fish or shellfish that was most resistant, 17 isolates in total. The antibiotic resistance profiles and the genotypes are given in the Table 5. Ten isolates were resistant to 11 antibiotics and susceptible to only colistin. Two isolates harboured all six β-lactamase genes tested. Other isolates showed high but varying levels of resistance to the tested antibiotics, and also harboured multiple β-lactamase genes.

Conclusions
The resistance patterns of E. coli observed in this study suggests anthropogenic sources of contamination of seafood. Release of raw or partially treated sewage into fishing areas is a major concern that requires the attention of the local regulatory bodies. Seafood can be a source of dissemination of antibiotic-resistant bacteria into the community. Incidence of MDR bacteria can also potentially jeopardise the international fish trade. In this regard, urgent measures are necessary to implement suitable policy to monitor the quality of coastal waters, ensure hygiene in retail fish markets and establish suitable testing methods to detect MDR bacteria in seafood.