Plasmid-Mediated Antibiotic Resistant Escherichia coli in Sarawak Rivers and Aquaculture Farms, Northwest of Borneo

Abstract

Background: The emergence of plasmid-mediated antibiotic resistance in Escherichia coli in water resources could pose a serious threat to public health. The study aims to investigate the dispersion of plasmid-mediated antibiotic-resistant E. coli from six rivers in Sarawak and two aquaculture farms in Borneo.

Methods: A total of 74 water samples were collected for the determination of their bacteria colony count. An IMViC test identified 31 E. coli isolates and tested their susceptibility against twelve clinically important antibiotics. The extraction of plasmid DNA was done using alkali lysis SDS procedures. Characteristics, including plasmid copy number, molecular weight size, resistance rate and multiple antibiotic resistance (MAR), were assessed.

Results: Our findings revealed that bacterial counts in rivers and aquaculture farms ranged from log 2.00 to 3.68 CFU/mL and log 1.70 to 5.48 cfu/mL, respectively. Resistance to piperacillin (100%) was observed in all E. coli; resistance to amoxicillin (100%) and ampicillin (100%) was observed in E. coli found in aquaculture farms; resistance to streptomycin (93%) was observed in E. coli found in rivers. All E. coli were resistant to ≥2 antibiotics and formed 26 MAR profiles, ranging from an index of 0.17 to 0.83, indicating that there are high risks of contamination. Some (48.4%) of the E. coli were detected with plasmids (1.2 to >10 kb), whereas 51.6% of the E. coli did not harbor any plasmids. The plasmid copy numbers reported were one plasmid (n = 7), two plasmids (n = 4), ≥ two plasmids (4). E. coli isolated from the Muara Tuang River showed the highest-molecular-weight plasmids. A statistical analysis revealed that there is no significant correlation (r = 0.21, p = 0.253) between the number of plasmids and the MAR index of the tested isolates.

Conclusion: The distribution of MAR in E. coli from rivers is higher compared to the aquaculture environment. Our study suggests that MAR in isolates could be chromosome-mediated. Our results suggest that riverbed sediments could serve as reservoirs for MAR bacteria, including pathogens, under different climatic conditions, and their analysis could provide information for public health concerns.

Keywords: Escherichia coli; aquaculture; drug resistant; extrachromosomal; plasmid; water source.

Figure 1

The antibiotic resistance rate (%) of Escherichia coli isolated from aquaculture farms and Sarawak Rivers against 12 antibiotics. AMC: amoxicillin, S: streptomycin, CIP: ciprofloxacin, K: kanamycin, CN: gentamicin, PRL: piperacillin, TE: tetracycline, AK: amikacin, AMP: ampicillin, NA: nalidixic acid, F: nitrofurantoin, C: chloramphenicol. Values represent the mean (%) of three replications. Vertical bars indicate the standard error of the means.

Figure 2

The percentage (%) of Escherichia coli isolated from 6 Sarawak rivers that showed Susceptible, Intermediate and Resistant against the 12 antibiotics tested.

Figure 3

The percentage (%) of Escherichia coli isolated from aquaculture farms that showed Susceptible, Intermediate and Resistant against the 12 antibiotics tested.

Figure 4

Multiple-antibiotic-resistance profile of E. coli from 6 Sarawak rivers and 3 aquaculture farms.

Figure 5

Agarose gel electrophoresis photograph of the estimated molecular weight size (kb) of the plasmid fragments in Escherichia coli isolated from Sarawak rivers and aquaculture farms. The estimated plasmid size was measured by comparing their band patterns obtained in agarose gel with linear DNA markers. Lane M, 1kb ladder; Lane 1, E. coli ATCC 25922; Lane 2, WF-S2B; Lane 3, WF-S3B, Lane 4, BT-S3.

Figure 6

Agarose gel electrophoresis photograph of the estimated molecular weight (1.1- >10 kb) of the plasmid fragments in Escherichia coli isolated from Sarawak rivers and aquaculture farms. The plasmid DNA band shown above the chromosomal DNA band was estimated to carry >10 kb molecular weight. Lane M, 1kb ladder; E. coli isolates (A) Lane 1, E. coli ATCC 25922; Lane 2, WC-S1A; Lane 3, SN-S2B; Lane 4, MT-S1A; Lane 5, SN-S1B; (B) Lane 1, AS-62(S); Lane 2, BK-RV1(S); Lane 3, AS-8(S); Lane 4, AS-NS(S)2; Lane 5, BK2-K4(S); Lane 6, AS-16(S); (C) Lane 1, AS-SD2(B); Lane 2, BK2-OLT(B); Lane 3, AS-16(B); Lane 4, BK2-OLT(S); (D) Lane 1, AS-R10(S); Lane 2, AS-62(B); Lane 3, BK2-K2(B); Lane 4, AS-R10(B); (E) Lane 1, BK2-OLT(S); Lane 2, BK2-OLT(B); Lane 3, AS-16(B); Lane 4, BK-RV2(S); (F) Lane 1, BT-S3B; Lane 2, WF-S3B; Lane 3, WF-S2B; Lane 4, SN-S2B; Lane 5, BK-RV2(S); Lane 6, SN-S1B; (G) Lane 1, WF-S3 B; Lane 2, WC-S3A P; Lane 3, WF-S1B; Lane 4, SN-S1B; Lane 5, AS-42(S); Lane 6, BT-S1B; (H) Lane 1, SN-S1A; Lane 2, AS-62(B); Lane 3, AS-R10(B); Lane 4, AS-16(B); Lane 5, WF-S1B; Lane 6, BK2-K2(B).

Figure 7

Locations of the study areas in Sarawak, Malaysia, northwestern Borneo (shown in the red box). (A) Locations of six sampling stations of rivers and (B) locations of three aquaculture farms.