Phenotypic and Genotypic Analysis of Beta-Lactamases in Escherichia coli Isolated from Fish

E. coli isolates were recovered from fish samples collected from local markets in Peshawar, Pakistan following standard protocols as described previously (De Vos, 2009). A total of fifty isolates were investigated in the study.

Phenotypic detection of ESBLs was carried using double disc synergy test. Amoxicillin-clavulanic acid disc was applied to the centre of Muller Hinton Agar (MHA) plate having bacterial lawn. The remaining discs of aztreonam, cefepime, cefotaxime and ceftazidime were applied around the central disc with 15-20 mm away. Samples were incubated overnight at 37˚C. Zone diameters were calculated and increase in zone towards the central disc was regarded as indication of ESBL production.

Metalo beta-lactamases were phenotypically analyzed by combine disc test. Two discs imipenem were placed about 25 mm apart onto MHA plate with bacterial lawn. One disc was applied with 5 µl 0.5 M EDTA (pH 8.0). the samples were incubated overnight at 37 ˚C. Inhibition zones were recorded and increase of ≥ 7 mm in inhibition zone around imipenem-EDTA was considered as positive result for MBL production.

For molecular detection of resistant genes, DNA was isolated according to the procedure of Wilson (1987). Quality was confirmed through gel electrophoresis. Three ESBL genes (blaCTX-M, blaSHV, blaOXA-10) and one MBL gene (blaNDM-1) were analyzed among the isolates using specific primers (Peerayeh et al., 2014; Shanthi et al., 2014). A 20 µl reaction mixture containing 4 µl 5x FIREPol Master Mix (Solis BioDyne, Cat. No. 04-12-00125), 1 µl each of gene specific forward and reverse primer, 1 µl DNA template and 13 µl molecular grade water was prepared. Previously reported amplification profiles were used for each gene (Peerayeh et al., 2014; Shanthi et al., 2014). The products of PCR were analysed on 1.5% agarose gel and compared with 100 bp plus DNA ladder (BIORON, Cat. No. 304105).

A total of 50 E. coli isolates were recovered from fish samples. Among the 50 isolates, 16% (n=4) were phenotypically confirmed as ESBLs producers. MBL-producers were not detected by phenotypic analysis. Molecular analysis showed the occurrence of two ESBL genes i.e. bla CTX-M and bla SHV. While bla OXA-10 gene was not detected. The occurrence of MBL gene bla NDM-1 was also confirmed among the isolates. The presence of bla CTX-M was confirmed by amplification of a 552 bp fragment corresponding to the gene. The presence of bla SHV was confirmed by the amplification of a 230 bp fragment specific to the gene while existence of bla NDM-1 was confirmed by the amplification of a gene specific fragment of 475 bp. The bla CTX-M gene was detected in 20 isolates (F6G, F7D, F7C, F11C, F14D, F13L, F14K, F13M, F13E, F13Q, F13F, F13D, F14Q, F13N, F14F, F7B, F6C, F9D, F7A and F11A). The blaSHV gene was present in 30 isolates (F14E, F13D, F9C, F14J, F14M, F14B, F14F, F13I, F14P, F13E, F7G, F9D, F6D, F6C, F14D, F13P, F14G, F14I, F13Q, F7I, F13O, F14K, F7A, F13A, F14O, F13N, F6A, F12A, F10D and F13C). The blaNDM-1 gene was detected in 3 isolates (F14G, F14F and F11C).

Recently, foodborne E. coli and other pathogenic members of the family Enterobacteriaceae are becoming a serious health risk as new strains are increasingly reported to possess multiple antibiotic resistance genes including ESBLs and MBLs. A study from Vietnam reported that nearly 30% of farmed fish and shrimp were contaminated with E. coli able to produce beta-lactamases (Nguyen et al., 2016). Another study from Vietnam reported 18.3% fish contamination with bacteria in local retail store (Le et al., 2015). In the same way in another study from Switzerland, the gut samples of fish were found to have 18.7% prevalence of ESBL or pAmpC-producing E. coli with two isolates having blaSHV-12 and one isolate contained blaCMY-2 gene (Abgottspon et al., 2014). In study in Saudi Arabia 51% fish samples had ESBL-producing E. coli (Elhadi and Aslmmann, 2015). In the current study, 16% E. coli isolates were phenotypically confirmed as ESBL producers. The beta lactamase gene blaCTX-M and its variants are the predominant with respect to geographical location. The blaCTX-M-1 gene has been reported from different parts of European countries (Coque et al., 2008), and blaCTX-M-8 and blaCTX-M-2 have been reported from South America (Egervärn et al., 2014) and Japan (Kawamura et al., 2014). In Vietnam, blaCTX-M-9 and blaCTX-M-1 have been frequently reported in fish and meat samples (Nguyen et al., 2016). In the current SHV-1 gene was the most prevalent (60%) while CTX-M gene was the second most abundant (40%) gene.

The increased prevalence of ESBL and MBL producing E. coli in fish could be due to overuse of antibiotics in aquaculture and due to disposal of domestic waste into aquatic habitats. Aquaculture is a comparatively new field where antimicrobial drugs are profoundly used, either by directly adding antibiotics to the fish food or to the water in fish farms. The indirect dissemination of antibiotic resistance genes from antibiotic-resistant bacteria from fish bodies to human pathogenic bacteria like E. coli and Salmonella has been reported in Aeromonas isolated from fish (Heuer et al., 2009; Cabello, 2006). The appearance of MDR strains of Vibrio cholerae which was the cause of Latin American outbreak in the year 1992 was another example where the spread of multi drug resistance occurred through seafood and water (Weber et al., 1994). Similar reports on outbreaks of MDR strains of Vibrio alginolyticus and Vibrio parahaemolyticus from farmed fish in Korea haven been documented (Oh et al., 2011). Increasing number antibiotic resistant Salmonella enterica in the United States and Europe has been proposed to have originated from aquaculture environment (Cabello, 2006; Briggs and Fratamico, 1999; Angulo, 2000; Yang et al., 2013).

The high prevalence of ESBL and MBL producing E. coli originating from fish shows the presence of a potential reservoir of pathogenic bacteria. These bacteria could spread to human population through food chain. Hence, proper surveillance system needs to be put in place to constantly monitor fish farming and related industries for existence of such resistant bacteria. Strict safety measures must be taken to monitor the points of contamination after harvesting the seafood to restrict the spread of multidrug resistant bacterial strains.

The authors have declared no conflict of interest.