Molecular Typing of β-Lactamase and Tetracycline Resistant Escherichia coli Strains Isolated from Imported Shrimp

Special Article - Escherichia coli

J Bacteriol Mycol. 2019; 6(3): 1102.

Molecular Typing of β-Lactamase and Tetracycline Resistant Escherichia coli Strains Isolated from Imported Shrimp

Khan S¹, Campbell M², Alotaibi K³, Smani D4, Khan A4, Sung K4, Khan S4 and Nawaz M4*

¹University of Arkansas, USA

²Hendrix College, USA

³Department of Biology, Jazan University, Kingdom of Saudi Arabia

4Division of Microbiology, National Center for Toxicological Research, FDA, USA

*Corresponding author: Mohamed Nawaz, Division of Microbiology, National Center for Toxicological Research, Jefferson, AR 72079, USA

Received: February 14, 2019; Accepted: March 28, 2019; Published: April 04, 2019


The misuse of antibiotics in commercial aquaculture may result in the selection of antibiotic-resistant bacteria. A study was undertaken to isolate and characterize the prevalence of β-lactam and tetracycline-resistant Escherichia coli from imported shrimp. All 55 strains of E. coli isolated from 207 shrimp samples were resistant to ampicillin, penicillin and tetracycline. These isolates were screened for 11 different β-lactam resistance genes by PCR. Oligonucleotide primers specific for the amplification of a 550-bp portion of the blaCTX-M gene amplified this gene from 31 of the 55 (56%) isolates. Oligonucleotide primers specific for the amplification of a 851-bp portion of the blaTEM gene amplified the genes from 9 (16%) of the isolates. Six (11%) were found to harbor both the 550-bp blaCTX-M and the 851-bp blaTEM genes. Template DNA were also screened for the presence of 6 different tetracycline resistance (tet) genes, PCR detected the presence tetB in a majority (39/55, 71.0%) of the isolates, followed by tetA (13.0%). Eleven percent contained both tetA and tetB genes. The PCR based Replicon Typing Method (PBRT) was used for typing plasmids from the 55 isolates by targeting the replicons of 15 major known plasmid families. Majority (44/55, 80.0%) of the isolates contained B/O plasmid replicon. Seven (12%) isolates also contained plasmids of the FIA family and 5 (9%) isolates contained both B/O and FIA plasmid families. The β-lactam and tetracycline resistant determinants were successfully transferred to E. coli J53 by conjugation. Our results indicate that imported shrimp may be a reservoir of the known β-lactam and tetracycline-resistance determinants.


Asian nations, such as Thailand, Vietnam, Indonesia, China, India and Bangladesh, currently produce nearly 80% of the world’s farmed shrimp [1-3]. These Asian nations earn more than $5 billion by exporting 1.3 billion of farm-raised shrimp. Intense aquaculture demands the implementation of modern technologies to enhance production and profits [4,5]. However, aquaculture production is affected by several bacterial diseases. Thus, large amounts of antibiotics are broadly used in aquaculture farming to promote growth and to retard the incidence and effects of diseases caused by overcrowded aquaculture ponds [1,2]. The excess use of antibiotics in aquaculture ponds may select bacteria resistant to multiple antibiotics [1,5]. Several public health agencies in the United States limit the presence of antibiotic residues in food-producing animals to decrease the prevalence of antibiotic-resistant microflora in food-producing ecosystems [6].

β-Lactam antibiotics (including penicillin derivatives, cephalosporins, monobactams and carbapenems) and tetracyclines are widely used for the treatment of urinary tract, respiratory tract, the intestines, certain inflammatory disorders and numerous other life threatening infections [7]. However, the widespread use of the same types of drugs in aquaculture ponds to prevent the outbreak of diseases may select bacteria resistant to these life-saving drugs and reduce the efficacy of these drugs in clinical treatment of infectious diseases [1,5,6].

Several mechanisms of resistance to β-lactam and tetracycline antibiotics have been reported [8-10]. Additional mechanisms, such as altered cell membrane permeability and overexpression of multidrug efflux pumps, also contribute to high-level bacterial resistance to these antibiotics. However, limited information is available on the occurrence and prevalence of β-lactam and tetracycline-resistant determinants in E. coli in imported shrimp. There is also a paucity of information on the types of plasmid replicon prevalent in aquaculture ecosystem. Such information is indispensable for understanding the epidemiological dynamics and to devise interventional strategies to curtail the dissemination of specific plasmid replicons to other ecosystems. In this report, we describe the occurrence and prevalence of different β-lactam and tetracycline resistance determinants and plasmid replicons in E. coli isolated from imported shrimp samples.

Materials and Methods

Bacterial strains: All the strains of E. coli used in this study were isolated from shrimp (Penaeus monodon) imported to the US. The isolation, characterization and identification of these isolates were described earlier [11]. All isolates were stored in Luria Broth (LB) containing 20% glycerol at -70°C and were grown overnight at 37°C in LB or on Trypticase Soy Agar (TSA) plates supplemented with 5% sheep blood.

Determination of antibiotic susceptibility and the minimum inhibitory concentration (MIC) of the isolates: The antibiotic susceptibility of each isolate was determined by disk diffusion assay [12] and was interpreted as per the criteria specified by the Clinical and Laboratory Standards Institute (CLSI). The MICs for the antibiotics (tetracycline, ampicillin and penicillin) were determined by broth dilution using Mueller-Hinton broth [13].

Genomic DNA extraction

Genomic DNA was extracted from cells grown overnight at 37°C with the QIAamp DNA Mini Prep Kit (Qiagen, Valencia, CA).

Detection of β-lactamase genes from template DNA

The presence of various β-lactam resistance genes in the template DNA was determined by PCR [14]. The primers used for the amplification of these genes are listed in Table 1. PCR amplification of the β-lactam resistance genes was carried out in a reaction volume of 25 μl using a PCR Kit (Applied Biosystems, Foster City, CA). The thermal cycling conditions consisted of an initial denaturation of 94°C for 2 min followed by 35 cycles of amplification. Each cycle consisted of 94°C denaturation for 30 s, annealing for 1°C below the lowest Tm of a given primer pair, and 72°C extension for 1 min. The amplified PCR products were maintained at 4°C. A reagent blank contained all the components of the reaction mixture except template DNA, for which sterile distilled water was substituted. The PCR products were subjected to electrophoresis on 1.2% agarose gels in 1 x Tris-Borate- EDTA (TBE) buffer, visualized with UV, and photographed using an Eagle Eye II gel documentation system (Stratagene, La Jolla, CA). A 100-bp DNA ladder (Thermo Fisher Scientific, Grand Island, NY) was used as the size standard.