3.1. Prevalence of E. coli O157and Antibiotic Susceptibility of Isolates
A total of 184 E. coli
isolates were obtained from 228 water samples and 112 diarrhoeal stool specimens. Twenty-one (11.4%) of these 184 isolates were found to be non-sorbitol-fermenting, out of which 20 (95.2%) were confirmed to be E. coli
O157 by slide agglutination. Table 1
shows the prevalence of Escherichia coli
O157 in surface waters and among diarrhoeal patients. Only five of the 228 water samples were positive for E. coli
O157, corresponding to an isolation rate of 2.2 %. Only six of the 112 stool specimens gave positive cultures of E. coli
O157, and hence a prevalence of 5.4%.
shows the antimicrobial susceptibility patterns of 184 E. coli
, isolated from clinical and water samples, using 10 antibiotics. Gentamicin, norfloxacin and chloramphenicol were the most active. While all of the clinical isolates were sensitive to both gentamicin and norfloxacin, and only 6.8% was resistant to chloramphenicol, only 2.1%, 4.9% and 5.4% of the aquatic isolates were, resistant to the three antibiotics, respectively. Also, of the 184 isolates, 154 (83.7%) and 123 (66.8%) were resistant to ampicillin and tetracycline, respectively, implying that ampicillin and tetracycline were the least active.
reveals the multidrug resistance (MDR) patterns of the 184 E. coli
isolates. MDR was higher amongst aquatic isolates than the clinical isolates. While 58 (60.4 %) of the aquatic strains showed resistance to ampicillin, tetracycline and nitrofurantoin, only 22 (25 %) of the clinical isolates gave similar results. Overall, higher numbers of resistant isolates were identified among the aquatic isolates. The highest levels of MDR observed were in two aquatic isolates: one was resistant to eight antibiotics and another to nine of the 10 antibiotics tested, including ampicillin, cefuroxime, chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin, nalidixic acid, norfloxacin and tetracycline.
shows that amongst the 20 E. coli
O157 isolates (14 aquatic and six clinical strains), the most active antibiotics were the fluoroquinolones and chloramphenicol. While none (0.0 %) was resistant to norfloxacin, three (15 %) showed intermediate susceptibility and 17 (85 %) were sensitive. For ciprofloxacin, while 18 (90%) were sensitive, only 2 (10%) were sensitive. Seventeen (85 %) were sensitive to chloramphenicol. Further, 16 (80 %) of the E. coli
O157 isolates were sensitive to each of cotrimoxazole and gentamicin. All (100 %) of the six clinical O157 isolates were sensitive to ciprofloxacin and chloramphenicol, while five (83.3 %) and three (50 %) were resistant to tetracycline and ampicillin respectively. Amongst the 14 aquatic isolates, while 10 (71.4%) were resistant to ampicillin and tetracycline, nine (64.3%) were resistant to nitrofurantoin and 13 (93%) to chloramphenicol.
3.2. Plasmid Studies
Thirty-five multidrug-resistant E. coli
isolates were screened for plasmids. This consisted of 23 aquatic and 12 clinical isolates, grouped further into 20 E. coli
O157 strains and 15 non-O157 strains. Table 5
shows that 22 (62.9%), of the 35, haboured plasmids (>2.1 Kb). The table further reveals that out of the 22 plasmid-containing isolates, only seven (JC17, JC15, LR 11, BS12, AS04, FD08 and FD07), marked with asterisks (*) are E. coli
O157 strains. All (100%) of the 15 non-O157 isolates contained plasmids. The resistance level observed in this 22 ranged from two-drug resistance (Isolate JC17) to nine-drug resistance (Isolate AS03).
All (100%) of the 22 plasmid-containing isolates were resistant to tetracycline, while only 10 (45.5%) were resistant to ciprofloxacin. In all, eight isolates contained two or more plasmids, giving a prevalence of 22.9%. Each of the isolates bearing multiple plasmids was resistant to at least four antibiotics. Interestingly, Isolate AS03 which showed resistance to nine different antibiotics contained only one plasmid.
Amongst the 20 E. coli O157 isolates, the DNA fingerprints revealed that only seven (35%) contained plasmids, out of which three (15%) contained more than one plasmid. One of the aquatic isolates (FD07) containing two plasmids was resistant to seven drugs including ampicillin, cefuroxime, ciprofloxacin, cotrimoxazole, nalidixic acid, nitrofurantoin and tetracycline. No clinical E. coli O157 isolate contained multiple plasmids.
Plasmid curing experiments were carried out to determine if the drug resistance observed in this study was plasmid-mediated. No plasmid bands were observed after the electrophoretic separation of crude DNA extracts from seven tetracycline-susceptible mutants. Loss of plasmids correlated with loss of resistance to antibiotics in cured strains selected on tetracycline (50 μg/mL)-nutrient agar plates. Loss of resistance to multiple antibiotics was also observed. Six (86%) of the tetracycline-susceptible mutant showed sensitivity to tetracycline and ampicillin.
The detection of E. coli
O157 at relatively high frequencies in these waters (stream, 1.7%; dam, 2.7%; river, 2.1%) and its occurrence heighten public health concern about these surface waters that are sources of raw water treated for drinking, recreation, irrigation and herd watering [25
]. Kubanni River, used by local farmers for the irrigation of commercial crops (tomatoes, lettuce, cabbage, onions, spinach, sugarcane etc), has been reported as unfit for fresh produce irrigation [26
]. The 5.4% prevalence of E. coli
O157 in children with diarrhoea suggests that the epidemiological situation in Nigeria had remained the same since 1994 [14
Comparison of antibiotic susceptibility in clinical and water isolates showed that higher levels of resistance existed in the aquatic isolates. This agrees with the findings of Idika [25
], who studied Vibrio cholerae
isolates during an outbreak of cholera in Lagos in 1997 and reported that the isolates from water were resistant to tetracycline and gentamicin whereas clinical isolates were susceptible. Our findings which are not enough to suggest any epidemiological link between the aquatic and clinical strains concur with that of Okoh and Igbinosa [26
]. They studied antibiotic susceptibility patterns of the Vibrio
isolates as well as the distribution of antibiotic resistance genes in the isolates and found that previous use of antibiotics in earlier outbreaks may be, in part, responsible for the extensive increase in antibiotics resistance. It is unknown whether the isolates responsible for earlier and recent epidemics are of the same clonal origin. The association between the development of resistance to ampicillin and tetracycline with large-scale use of antibiotics for treatment and prophylaxis of diarrhoea is well recognized Idika [25
]. Ash et al.
] also reported high levels of resistance in gram-negative bacteria in rivers in the United States.
All (100%) of the 22 plasmid-containing isolates were resistant to tetracycline. This is worrisome considering that tetracycline is a first line drug in Nigeria, and as in most developing countries, people with gastrointestinal infections readily purchase it across the counter for self-medication.
From the results, it appears that plasmids are ubiquitous in bacterial population in the waters. Different plasmids (2–3) often coexisted in the same host cell. This agrees with the finding of Fujita et al.
] who isolated 45 antibiotic resistant bacteria from wastewater samples and detected 31 plasmids in 14 of 45 antibiotic resistant strains with 10 carrying multiple (2–4) plasmids. The demonstration of high-molecular-weight plasmids in the isolates, and the finding that the expressed multi-drug resistance (MDR) was plasmid-mediated is significant. McPherson and Gealt [3
] showed that several enteric bacterial strains isolated from wastewater samples and possessing both antibiotic resistance and high molecular-weight plasmids could transfer their resistance to other recipient bacteria. The transmissibility of resistance (R)-genes and plasmids poses public health risk, considering the vast potential of hosts presented by microbial populations in the gut and water environment. This risk could be heightened if R-genes are disseminated across geographic borders by travellers or by a river continuum, like Kubanni that traverses different communities within the study area.
Mutant cells obtained by SDS treatment of tetracycline-resistant isolates were susceptible to tetracycline suggesting that a part or all of their resistance genes must have been deleted by SDS action. Loss of plasmids correlated with loss of resistance to ampicillin, chloramphenicol, ciprofloxacin, norfloxacin and tetracycline in 6 (86%) of the mutants. It therefore appears that the observed multi-drug resistance to these antibiotics was plasmid-mediated. This agrees with previous findings.
Ash et al.
] had in a study of the antibiotic resistance of gram-negative bacteria in rivers in the United States demonstrated that resistance to ampicillin and other drugs (including ciprofloxacin, tetracycline, chloramphenicol, kanamycin and streptomycin) was plasmid-mediated. Interestingly, two mutants now resistant to cotrimoxazole originated from wild-type E. coli
O157 isolates that were sensitive to the same drug. This could be attributed to possible mutation affecting chromosomal genes in the course of the curing experiment [29