Abstract: In the present study, antibiotic susceptibility test was performed using the disc diffusion method and the results interpreted using the Clinical and Laboratory Standards Institute guidelines. A total of 45 Escherichia coli isolates were screened against nine different antibiotics. Overall, 34.57% of the Escherichia coli isolates were resistant, 7.16% were intermediate and 58.27% were susceptible. Resistance to vancomycin (88.89%) and erythromycin (68.89%) was high. Susceptibility to ciprofloxacin (95.56%), amoxycillin/clavulanic acid (86.67%), suphamethoxazole/ trimethoprim (82.22%) and gentamicin (75.56%) was also high. The Escherichia coli isolates also exhibited 25 antibiotic resistant patterns with the pattern VaE (vancomycin and erythromycin) and VaCCro (vancomycin, chloramphenicol and ceftriaxone) being the commonest (each exhibited by five different isolates). Multiple Antibiotic Resistance index (MAR index) ranged from 0.11-0.78. Resistance to seven (MAR index of 0.78) and five (MAR index of 0.56) different antibiotics was exhibited by 1 and 3 isolates, respectively. Some Escherichia coli isolates from different sources did exhibit the same resistance pattern. This study established the fact that Escherichia coli from meat and it related samples in Techiman Municipality were resistant to some antibiotics. Therefore, the use of antibiotics in the treatment of animals in the Municipality ought to be checked and controlled to prevent more isolates from becoming resistant. To the best of author’s knowledge, this is the first report on the antibiotic resistance of Escherichia coli isolated from beef and its related samples in Techiman Municipality of Ghana.
INTRODUCTION
Escherichia coli are gram negative, facultative anaerobic and non-spore forming bacteria commonly found in the gastrointestinal tract of humans, farm animals, pests and wild animals (Anonymous, 2012; Malik et al., 2013; Kumar et al., 2013, 2014; Anita et al., 2014; CDC., 2014a). Escherichia coli cells are rod-shaped and are about 2.0 μm long and 0.25-1.0 μm in diameter, with a cell volume of 0.6-0.7 μm3 (Kubitschek, 1990). They also ferment glucose or lactose and are members of the Enterobacteriaceae family (CDC., 2014a). Most Escherichia coli strains are harmless and form part of the normal flora of the gastrointestinal tract of animals (Aarestrup et al., 2008; Anonymous, 2012). Eckburg et al. (2005) reported that Escherichia coli together with other facultative anaerobic constitute about 0.1% of gut flora. Beneficial Escherichia coli can produce vitamin K2 for the host while pathogenic strains can cause bloody diarrhoea, anaemia, urinary tract infection, meningitis, peritonitis and even death (Bentley and Meganathan, 1982; Von Baum et al., 2005). Faeco-oral transmission is one of the major routes through which pathogenic strains found their way into food or water and consequently cause disease (Schroeder et al., 2002; Aarestrup et al., 2008; Anita et al., 2014).
Escherichia coli have been isolated from beef, it related samples, water sources, humans, etc. have been implicated in foodborne outbreaks (Kilic et al., 2007; Adzitey et al., 2011, 2012a, 2013, 2014; Islam et al., 2011; Bekele et al., 2014; CDC., 2014b; Carnot et al., 2014). Adzitey et al. (2014) identified Escherichia coli in beef sold in the Yendi Municipality of Ghana. The prevalence of Escherichia coli O157:H7 in beef samples in Addis Ababa, Ethiopia was 13.3% (17/128) (Bekele et al., 2014). In USA, CDC. (2014b) reported an outbreak of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7) infections resulting from the consumption of contaminated ground beef. Twelve people were infected and 58.0% of them were hospitalized (CDC., 2014b). Antibiotics play very important role in decreasing diseases, illness and/or death associated with bacterial infections in humans and animals. Nonetheless, the use of antibiotics as growth promoters and therapeutic purposes have been the major driving force behind the emergence and spread of drug-resistance bacteria among pathogenic and non-pathogenic bacteria strains (Schroeder et al., 2002; Aarestrup et al., 2008; Adzitey et al., 2012b). According to FDA (2014), surveillance data reveal that resistance in Escherichia coli is consistently the highest for antimicrobial agents that have long been in use in human and veterinary medicine.
Data on the antibiotic resistance of Escherichia coli in Ghana is scarce and in the Techiman Municipality of Ghana such data is unavailable. Therefore, this work report for the first time on antibiotic resistance of Escherichia coli isolated from beef and it related samples in Techiman Municipality of Ghana.
MATERIALS AND METHODS
Sources of Escherichia coli isolates: Forty five (45) Escherichia coli isolated from beef and its related samples between May 2013 and June 2014 in the Techiman Municipality of Ghana was used for this study. The Escherichia coli isolates were obtained from beef (n = 17), tables (n = 14) and knives (n = 14).
Antimicrobial susceptibility of Escherichia coli: The disk diffusion method of Bauer et al. (1966) was used to determine the antibiotic resistance of 45 Escherichia coli against the following antibiotics; amoxycillin/clavulanic acid (Amc) 30 μg, chloramphenicol (C) 30 μg, gentamicin (Cn) 10 μg, ceftriaxone (Cro) 30 μg, ciprofloxacin (Cip) 5 μg, erythromycin (E) 15 μg, suphamethoxazole/trimethoprim (Sxt) 22 μg, tetracycline (Te) 30 μg and vancomycin (Va) 30 μg. The disks were purchased from Oxoid Limited, Basingstoke, UK. Pure cultures of Escherichia coli were grown overnight in Tryptic Soy Broth (TSB) (Oxoid Limited, Basingstoke, UK) at 37oC and the concentration adjusted using sterile TSB until a 0.5 McFarland turbidity was attained. One hundred microliter of the culture was then swabbed onto Mueller Hinton agar (Oxoid Limited, Basingstoke, UK) using a sterile cotton swab. Three antimicrobial disks were placed on the surface of the agar plate at a distance to avoid overlapping of inhibition zones. The plates were incubated at 37°C for 16-18 h and the results were interpreted as sensitive, intermediate, or resistant according to Clinical and Laboratory Standards Institute guidelines for (CLSI., 2006). The Multiple Antibiotic Resistance (MAR) index was calculated and interpreted according to Krumperman (1983) using the formula: a/b, where ‘a’ represents the number of antibiotics to which a particular isolate was resistant and ‘b’ the total number of antibiotics tested.
RESULTS AND DISCUSSION
The antimicrobial susceptibility of 45 Escherichia coli were determined against 9 antimicrobial agents and the results are presented in Table 1. The overall resistance, intermediate and susceptibility was 34.57% (140/405), 7.16% (29/405) and 58.27% (236/405), respectively. A large percentage of the Escherichia coli were resistant to vancomycin (88.89%) and erythromycin (68.89%) but susceptible to ciprofloxacin (95.56%), amoxycillin/clavulanic acid (86.67%), suphamethoxazole/trimethoprim (82.22%) and gentamicin (75.56%). Intermediate resistances were observed for all the antibiotics except suphamethoxazole/trimethoprim, vancomycin and tetracycline. Intermediate resistance refers to those Escherichia coli species that were not clearly resistant or susceptible. It has been suggested in clinical diagnoses that patients with intermediate results can be given a higher dosage of antibiotics (Lorian, 2005). Organisms that exhibit intermediate resistance also have the tendency to easily become resistant (Adzitey et al., 2012b). The use of antibiotics in the treatment of diseases and as growth promoters in farm animals, use of antibiotics for treating humans and other factors have been linked to the development of resistant microorganisms (Krumperman, 1983; Schroeder et al., 2002; Aarestrup et al., 2008; Adzitey et al., 2012b). Resistant Escherichia coli species can contaminate carcasses, processing equipment and other foods which pose a risk for public and animal health.
Table 2 shows the antibiotic resistance profile and Multiple Antibiotic Resistance (MAR) index of individual Escherichia coli from different sources. The Escherichia coli exhibited 25 antibiotic resistant patterns with MAR index ranging from 0.11-0.78. The majority of the Escherichia coli (14 isolates) were resistant to three antibiotics (MAR index of 0.33), followed by resistant to four antibiotics (13 isolates; MAR index of 0.44), resistant to two (10 isolates; MAR index of 0.22), resistant to one antibiotic (4 isolates; MAR index of 0.11), resistant to 5 antibiotics (3 isolates; MAR index of 0.56) and resistant to seven antibiotics (1 isolates; MAR index of 0.78). The resistance pattern VaE and VaCCro were the commonest and was exhibited by 10 (5 each) different Escherichia coli isolates. One Escherichia coli isolate was resistant to 7 different antibiotics and exhibited a resistant pattern of VaSxtAmcCTeCnE. Of the 3 Escherichia coli isolates that were resistant to five different antibiotics, 1 each exhibited the pattern VaSxtCTeE, VaCipTeCnE and VaAmcCCroE.
Studies on the antibiotic resistance of Escherichia coli isolates in Ghana are limited and the few studies available have also concentrated on human isolates. George et al. (2012) determined the antibiotic resistance patterns of Escherichia coli isolated from patients in selected hospitals in Kumasi, Ghana and reported that Escherichia coli isolates (28.6-46.4%) were resistant to gentamicin, ciprofloxacin and ceftriaxone while 14.4-47.4% gave intermediate responses. This study found 13.33% resistance to gentamicin, 2.22% resistance to ciprofloxacin and 26.67% resistance to ceftriaxone but intermediate responses were lower in this study (2.22-11.11%).
| Table 1: | Percentage antibiotic resistance of Escherichia coli isolated from meats and it related samples |
| *n: No. of resistant Escherichia coli, S: Susceptible, I: Intermediate, R: resistant, Amc: Amoxycillin/clavulanic acid, C: Chlorofloxacin, Cip: Ciprofloxacin, Cro: Ceftriaxone, Cn: Gentamicin, E: Erythromycin, Sxt: Suphamethoxazole/trimethoprim, Te: Tetracycline, Va: Vancomycin | |
| Table 2: | Antibiotic resistance profile and multiple antibiotic resistance index of individual Escherichia coli from different sources |
| Amc: *Amoxycillin/clavulanic acid 30 μg, C: Chloramphenicol 30 μg, Cip: Ciprofloxacin 5 μg, Cro: Ceftriaxone 30 μg, Cn: Gentamicin 10 μg, E: Erythromycin 15 μg, Sxt: Suphamethoxazole/trimethoprim 22 μg, Te: Tetracycline 30 μg, Va: Vancomycin 30 μg, MAR: Multiple antibiotic resistance | |
In Accra, Ghana Namboodiri et al. (2011) reported that thirteen (52%) of 2006 and 2007 human Escherichia coli isolates and 10 (66.7%) of 2008 human Escherichia coli isolates were resistant to ciprofloxacin. The proportion of isolates resistant to each antimicrobial in 2008 was significantly greater than those seen in 2006 (Namboodiri et al., 2011). Anonymous (2015) analysed Escherichia coli isolated from hospitalized patients in Ghana for their sensitivity to antibiotics and reported that 82 and 75% were resistant to tetracycline and chloramphenicol, respectively. Resistance to tetracycline (44.44%) and chloramphenicol (44.44%) were lower in this study.
In other countries such as Canada, Carson et al. (2008) reported that Escherichia coli isolated from 29 volunteer beef farms in Ontario were all susceptible to ceftriaxone and ciprofloxacin. This work found some resistances to ceftriaxone and ciprofloxacin but susceptibility to ciprofloxacin was very high (95.56%). In USA, FDA. (2011) reported that Escherichia coli isolated from ground beef in 2011 were resistant to tetracycline (17.7%), trimethoprim-sulfamethoxazole (2.3%), chloramphenicol (1.4%), gentamicin (0.5%), amoxicillin-clavulanic acid (0.5%), ceftriaxone (0.5%) and ciprofloxacin (0.0%). Comparatively, this study found higher resistances to tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, gentamicin, amoxicillin-clavulanic acid, ceftriaxone and ciprofloxacin; even though resistance to amoxicillin-clavulanic acid (4.44%) and ciprofloxacin (2.22%) was low in this study (Table 1). In Bangladesh, Escherichia coli isolated from urinary tract infection patients were resistant to tetracycline (59%), ceftriaxone (5.5%), ciprofloxacin (11.5%) and gentamycin (8.1%) (Bhowmick and Rashid, 2004). Schroeder et al. (2002) also in the USA indicated that the prevalence of resistance among human Escherichia coli O157:H7 isolates was similar to that among cattle isolates for chloramphenicol (0 versus 1%), tetracycline (7 versus 11%) and amoxicillin-clavulanic acid (0 versus 1%). Furthermore, all Escherichia coli O157:H7 isolates, regardless of the source of isolation, were susceptible to ceftriaxone, gentamicin, ciprofloxacin and trimethoprim-sulfamethoxazole.
The antibiotic resistances reported in this study were either different from or similar to reports by other researchers in Ghana and other countries. These differences and similarities are due to differences in samples examined, differences in year/time of sampling, sampling procedure employed, breakpoints used and the level of usage of antibiotics in animal farming and human therapeutic purposes (Schroeder et al., 2002; Carson et al., 2008; Namboodiri et al., 2011; FDA., 2011). A wide variety of antibiotics have been suggested to be used in the treatment of infections caused by Escherichia coli. Pitout (2012) reported that the β-lactams, fluoroquinolones, aminoglycosides and trimethoprim-sulfamethoxazole are often used to treat community and hospital infections due to E. coli. β-lactams disrupt cell wall synthesis by binding to and inhibiting the penicillin-binding proteins essential for transpeptidation and carboxypeptidation reactions in cell wall peptidoglycan synthesis (Pitout, 2015). Fluoroquinolones interfere with DNA supercoiling and promote DNA gyrase-mediated double-stranded DNA. The aminoglycosides bind irreversibly to the 50S subunit of the 70S bacterial ribosomes (Pitout, 2015). Pitout (2015) also indicated that sulfonamides and trimethoprim interfere with bacterial folic acid synthesis by inhibiting tetrahydropteric acid syntheses and dihydrofolate reductase, respectively. Madappa (2014) showed that Escherichia coli meningitis, pneumonia and cholecystitis/cholangitis can be treated with third-generation cephalosporins such as ceftriaxone. Escherichia coli pneumonia can also be treated with fluoroquinolones and antimicrobials, known to be useful in cases of traveler's diarrhea include trimethoprim/sulfamethoxazole and fluoroquinolones (Madappa, 2014). In this study, most of the Escherichia coli isolates were susceptible to ciprofloxacin (95.56%), amoxycillin/clavulanic acid (86.67%), suphamethoxazole/trimethoprim (82.22%) and gentamicin (75.56%). Therefore, ciprofloxacin (fluoroquinolones) can be the first antibiotic of choice for treating Escherichia coli infection caused by the consumption of beef in the Techiman Municipality of Ghana. In the absence of ciprofloxacin, amoxycillin/clavulanic acid may be used before suphamethoxazole/trimethoprim and gentamicin.
CONCLUSION
In conclusion, Escherichia coli isolates from beef and its related samples exhibited varying resistances to antibiotics. Averagely, 58.27% were susceptible, 7.16% were intermediate and 34.57% were resistant. High susceptibility was observed for ciprofloxacin, amoxycillin/clavulanic acid, suphamethoxazole/trimethoprim and gentamicin while high resistances were observed for vancomycin and erythromycin. Multiple antibiotic index ranged from 0.11-0.78 (that is resistant to 1-7 different antibiotics). Majority of the Escherichia coli isolates were resistant to three antibiotics, but the resistant pattern, VaE and VaCCro were the commonest. There is the need to control the use of antibiotics in animal farming and treatment of humans in Techiman Municipality of Ghana to control the incidence of increasing multidrug resistant Escherichia coli isolates. The findings of this study can serve as baseline information for the antibiotic resistance of Escherichia coli isolated from meat samples in Ghana to monitor trends in the future.