Plasmid profile of antibiotic resistant Escherichia coli isolated from chicken intestines.
Fifty-seven strains of Escherichia coli from chicken intestines were isolated and tested against 15 antibiotics by the disk diffusion method. All strains were resistant to at least one of the tested antibiotics. Resistance to 12 antibiotics was the maximum observed. 26.3% of the isolates exhibited multiple resistances to 50% of the tested antibiotics. Most of the isolates were resistant to tetracycline and nalidixic acid. R-plasmids were extracted and separated by agarose gel electrophoresis for profiling. Plasmid profiling of antibiotic resistant Escherichia coli isolates revealed that the isolates contained various size R-plasmids. Although some strains exhibited different antibiotic resistance patterns, some of their plasmids had similar migration patterns on agarose gel electrophoresis. Multiple resistances are conferred by R-plasmids of different sizes. The high prevalence of antibiotic resistance conferring plasmids observed in this study may be due to the increasing widespread use of antibiotics.
Antibiotics have helped in reducing the diseases in the poultry farms. However, there is a growing awareness of public health concerns associated with the use of antibiotics (Rice et al.., 1995). Although antibiotic use is under national regulations of Oman, farmers still overuse antibiotics. Carraminana (et al.., 2004) reported that 40% of the antibiotics produced in the United States were used in stock feeds. The widespread use of various antibiotics for treating chicken infections has created for antibiotic resistant bacterial strains (Chee-Sanford et al.., 2001, Sackey et al.., 2001). Bacterial isolates obtained by Carraminana et al.. (2004) from a poultry slaughter house in Spain had high percentages of resistance to many antibiotics such as neomycin (53.4%), tetracycline (21.8%), and streptomycin (11.3%). Saenz et al.. (2000) reported a high frequency of resistance to ampicillin (65.7%), gentamicin (22.2%), and amikacin (21.6%) in bacterial strains isolated from animals.
Multiple antibiotic resistant strains can be transported from animals to humans by food (Cordano and Virgilio, 1996). Linton et al.. (1977) reported that multiple resistant bacterial strains were transmitted to humans by raw meat and milk. A poultry feces is a potential source of antibiotic resistant bacteria. If released into the environment, resistant strains may contaminate water and food sources and can be a potential threat to human health (Chee-Sanford et al.., 2001). Escherichia coli strains isolated from sewage treatment plants were reported to be resistant to various antibiotics (Osterblad et al.., 2000 and Reinthaler et al., 2003).
In Saudi Arabia, Escherichia coli isolated from chicken intestines were found to be resistant to many antibiotics such as ampicillin, chloramphenicol, gentamycin, tetracycline, trimethoprim and sulphamethoxazole (Al-Ghamdi et al.., 1999). Mirza et al.., (2000) reported that antimicrobial resistance was transferable from Salmonella spp to Escherichia coli as well as between other members of the intestinal normal flora.
Plasmids are a major mechanism for the spread of antibiotic resistant genes in bacterial populations (Smalla et al.., 2000). Conjugation occurs by F-plasmids that can transfer genes encoded for multiple resistance and mobilize other non-conjugative plasmids to host cells (Saxena et al., 1984). Multiple resistance genes are harbored on R-plasmids some of which are conjugative (Elwell and Falkows, 1980). Escherichia coli has been reported to transfer the antibiotic resistant genes to enteric pathogenic and normal flora bacteria such as Salmonella spp and Proteus spp (Platt et al.., 1986). The objective of this study was to investigate plasmid profile of antibiotic resistant Escherichia coli isolated from chicken intestines.
MATERIALS AND METHODS
Twenty-eight chicken intestines were collected from different local slaughter houses. Escherichia coli strains were isolated according to standard methods (Sonnenwirth and Jarett, 1980). The isolates were identified biochemically by API system (API Analytical Products, New York, NY). Forty-seven Escherichia coli isolates were tested for susceptibility to antibiotics following the disk diffusion method (Bauer et al.., 1966). Fifteen antibiotics were used: Amikacin (AK) 30 [micro]g, Ampicillin (AMP) 10 [micro]g, Carbenicillin (CB) 100 [micro]g, Cephotaxin (CTX) 30 [micro]g, Chloramphenicol (CM) 30 [micro]g, Gentamycin (GM) 10 [micro]g, Kanamycin (KM) 30 [micro]g, Minocylin (MH) 30 [micro]g, NAlidixic Acid (NA) 30 [micro]g, Neomycin (NM) 30 [micro]g, Sulphamethoxazole (SMX) 30 [micro]g, Streptomycin (SM) 10 [micro]g, Tetracycline (TE) 30 [micro]g, Tobramycin (TOB) 10 [micro]g, and Trimethoprim (TMP) 5 [micro]g. Inhibition zone diameters were measured after 24 hours of incubation at 37[degrees]C. Antibiotic resistance patterns were recorded. A standard Escherichia coli (ATCC 10536) was used as a control.
Plasmids were extracted using a High Pure Plasmid Isolation Kit (Roche, 2003). The extracted plasmids were separated by agarose gel electrophoresis for their profiling. Gel electrophoresis was carried out on 0.7% agarose grade gel at 100 v for 2h (Sambrook et al, 1989).
Forty-seven Escherichia coli strains were isolated from twenty-eight chicken intestines and tested against 15 antibiotics. All strains were resistant to at least one antibiotic (Table 1; Fig. 1). Nineteen percent of the isolates (9 out of 47 isolates) were resistant to four tested antibiotics. Forty-five Escherichia coli isolates were resistant to 5-7 antibiotics. The broadest range of drug resistance was up to 12 antibiotics (2.1%).
Figure 2 also shows the antibiotic resistance patterns of the isolates. Most of the isolated strains shared resistance to four antibiotics, namely kanamycin, nalidixic acid, streptomycin, and tetracycline and therefore exhibited a common resistance pattern of KMNASMTE. The highest frequency was with tetracycline (97.9%) followed by NA (78.7%), SM (68.1%) and KM (59.6%). None of the strains were resistant to AK and CTX (Fig. 2).
A plasmid occurrence rate of 100% (47 out of 47) was observed. Plasmids analyses revealed that all resistant strains harbored 1-5 small size plasmids with molecular weight (m.w) in the range 2.9-66 kilo base (kb) (Table 1). Fifteen strains (31.9%) had one plasmid, 13 (27.7%) had two plasmids, 9 had three plasmids (19.1%), 8 strains had four plasmids (17%) and 2 (4.3%) harbored five plasmids. In general, strains resistant to one antibiotic contained one plasmid. However, strain 9 resistant to TE contained two plasmids (58 and 26 kb). Also, some of the isolates were resistant to seven antibiotics, strain 12, and strain 23 resistant to 9 antibiotics but contained 1 plasmid each, 3.3 kb and 43 kb respectively.
Reinthaler et al.., (2003) found that most Escherichia coli strains from sewage exhibited multiple resistance to antibiotics. Multiple resistance was reported to be more common than resistance to single antibiotics (Caudry and Stanisich, 1979). It was also reported that a high percentage of Escherichia coli (86.5%) isolated from avian feces were resistant to one or more antibiotics (Tabatabaei and Nasirian, 2003). In this study, all of the isolates were resistant to at least one antibiotic. Tetracycline resistance was also observed among Escherichia coli isolates and has been frequently reported in poultry products (Sackey et al., 2001). Tabatabaei and Nasirian, 2003 reported that 94% of Escherichia coli isolates from chickens were resistant to TE and 46% were resistant to KM. Their findings are similar to our results. Kariuki et al., (1999) reported that TE is one of the broad-spectrum antibiotics that are available in feed supplements, and its improper use led to the development of multiple antibiotic resistances. In the United States, resistance to TE increased from 9% in 1980 to 24% in 1990 (Winokur et al., 2000). Hofacre et al.., (2002) reported that 90% of Escherichia coli poultry isolates were resistant to TE. In Jamaica, 82.4% of Escherichia coli isolates were resistant to tetracycline (Miles at al., 2006). Reinthaler et al., (2003) showed that resistance to TE can be transferred into the environment.
The percentage resistance to NA (78.8 %) in our study was higher than that reported in Malaysia (13%) (Radu et al., 2001) and the United States (37 %) (Johnson et al.., 2003). In the present study, Escherichia coli strains were found to be resistant to CM and GM, whereas in Sweden, Ronner et al.., (2004) found that chicken isolates were not resistant to CM and GM. Aja et al., (2002) reported that some of their isolates were resistant to AK, but our study showed that our strains were not resistant to AK.
The high rates of resistance found in this study can be explained by the wide spread use of antibiotics in Oman for prophylaxis and for treatment in poultry farms. The improper and unnecessary use of antimicrobial drugs in man also promotes development of resistant strains with R-plasmids. Linton et al.., (1977) reported that both pathogenic and non-pathogenic strains resistant to drugs may be transported from animals to humans via food. Such strains act as an important source for in vivo transmission of R-plasmids to drug sensitive strains in the animal intestine mainly through conjugation (Platt et al., 1986). A great similarity between the plasmids of Enterobacteriaceae isolated from animals and humans has been observed. Other workers reported that transmission of resistance plasmids of Escherichia coli from poultry to human intestines commonly occurs (Tabatabaei and Nasirian, 2003).
The plasmid DNA analysis of the strains in this study showed that the size of the plasmid DNA varied. Although some strains were resistant to only one antibiotic, they had more than one plasmid while others containing I or 2 plasmids were resistant to a large number of antibiotics. Al-Bahry (2000) reported similar findings. In his study, plasmid DNA analysis of the 28 Salmonella strains showed that the size of the plasmid DNA ranged from 3.1 kb to 32 kb. A study by Son et al., (1997) on isolates from fish revealed a similar size range of R plasmids (3 to 63.4 kb).
Nine strains (19%) were found to harbor 54 kb plasmids. Icgen et al., (2002) reported that a common 54 kb plasmid was harbored by 55% of local isolates of Enterobacteriaceae from Turkey. Al-Bahry (2000) reported that most strains associated with non-human sources were found to harbor larger plasmids while most human strains have relatively much smaller plasmids with sizes 10.8, 9, 4.7 and 6.2 kb pairs, yet they were resistant to a larger number of antibiotics. This suggests that not all antibiotic resistance genes are located in plasmids. Some of the genes conferring resistance may be located on bacterial chromosome. Aja et al., (2002) in their study of Vibrio strains isolated from cultured shrimps reported that some strains were resistant to four antibiotics, others were resistant to two antibiotics and all contained one plasmid of 21.2 kb pair. They suggested that resistance to antibiotics could be encoded in some strains in plasmids and in others in the chromosomes.
It is well established that antibiotic pressure supports resistant strains and eliminates sensitive strains. The greater the overuse of antibiotics, the more the elimination of the sensitive strains allowing resistant strains to dominate. It is also true that resistant strains are outcompeted by sensitive strains when antibiotic pressure is removed from the environment. Thus, steps must be taken to control the overuse of antibiotics in Oman as well as in other developing countries.
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Saif N. Al-Bahry, Basma M. Al-Mashani, Abdulkadir E. Elshafie, N. Pathare and Asila H. Al-Harthy
Department of Biology, College of Science, Sultan Qaboos University. P.O.Box 36, Al-Khodh, P.C. 123, Sultanate of Oman.
Correspondence: Al-Bahry, Saif (firstname.lastname@example.org)
Table 1: Antibiotic resistance patterns and plasmid contents of Escherichia coli isolates. No of antibiotics Isolate E. coli isolates No of number Antibiotic resistance pattern resistant plasmid 1 KMNMSMTE 4 3 2 KMNANMSMTE 5 4 3 KMNANMTE 4 2 4 KMNANMSMTE 5 4 5 KMNANMTE 4 4 6 GMKMNANMSMTE 6 1 7 KMNANMSMTE 5 1 8 CMNASMTE 4 1 9 TE 1 2 10 CMMHNASMXSMTETMP 7 3 11 AMPCBCMKMMHNASMXTETMP 9 4 12 AMPCBNASMXSMTETMP 7 1 13 CMMHNASMXSMTETMP 7 2 14 KMNMSMTE 4 1 15 AMPCBCMKMMHNASMXTETOBTMP 10 3 16 KMMHNASMXTETOBTMP 7 3 17 NASMXTETMP 4 2 18 AMPCBNATE 4 1 19 AMPCBCMKMNANMSMXSMTETMP 10 4 20 SMTETOB 3 1 21 KMNMTE 3 3 22 CMKMNANMSMTE 6 2 23 AMPCBKMNANMSMXSMTETMP 9 1 24 NASMXSMTETMP 5 2 25 KMNMSMTE 4 5 26 NASMTE 3 5 27 KMMHNANMSMXTETMP 7 2 28 AMPCBCMKMMHNANMSMXSMTETMP 11 2 29 AMPCBCMGMKMMHNASMXTETOBTMP 11 4 30 AMPCBCMGMKMMHNASMXSMTETOBTMP 12 3 31 AMPCBCMKMMHNANMSMXSMTETMP 11 3 32 TE 1 1 33 NATE 2 3 34 KMNANMSMTETMP 6 3 35 KMNANMTE 4 4 36 KMNANMSMXSMTETMP 7 4 37 KMMHNANMSMTE 6 1 38 KMMHNANMSMTE 6 1 39 NA 1 2 40 AMPCBNASMTE 5 1 41 AMPCBCMNMSMTE 6 1 42 NASMXSMTETOBTMP 6 1 43 CMMHNASMXSMTETMP 7 1 44 CMKMNANMSMXSMTETMP 8 2 45 AMPCBCMKMNANMSMXSMTETOBTMP 11 2 46 AMPCBMHSMTE 5 2 47 AMPCBMHSMTE 5 2 Isolate number Antibiotic resistance pattern Plasmid m.w. (K.b) 1 KMNMSMTE 66,48,22 2 KMNANMSMTE 61,51,29,14.5 3 KMNANMTE 26,11 4 KMNANMSMTE 66,48,22,8.8 5 KMNANMTE 66,48,26,8.8 6 GMKMNANMSMTE 51 7 KMNANMSMTE 51 8 CMNASMTE 66 9 TE 58,26 10 CMMHNASMXSMTETMP 31,10.5,2.9 11 AMPCBCMKMMHNASMXTETMP 61,12,7.2,3.3 12 AMPCBNASMXSMTETMP 3.3 13 CMMHNASMXSMTETMP 61,31 14 KMNMSMTE 6 15 AMPCBCMKMMHNASMXTETOBTMP 45,28,11.5 16 KMMHNASMXTETOBTMP 45,28,11.5 17 NASMXTETMP 58,43 18 AMPCBNATE 14.5 19 AMPCBCMKMNANMSMXSMTETMP 66,40,16.5,6.3 20 SMTETOB 40 21 KMNMTE 45,16.5,8.2 22 CMKMNANMSMTE 54,11.5 23 AMPCBKMNANMSMXSMTETMP 43 24 NASMXSMTETMP 51,12.5 25 KMNMSMTE 45,38,26,22.6.8 26 NASMTE 54,38,26,12,3.8 27 KMMHNANMSMXTETMP 54,10.5 28 AMPCBCMKMMHNANMSMXSMTETMP 54,3.5 29 AMPCBCMGMKMMHNASMXTETOBTMP 51,33,22,4 30 AMPCBCMGMKMMHNASMXSMTETOBTMP 54,36,26 31 AMPCBCMKMMHNANMSMXSMTETMP 54,36,6 32 TE 7.8 33 NATE 40,33,12.5 34 KMNANMSMTETMP 54,33,8.2 35 KMNANMTE 58,43,28,16.5 36 KMNANMSMXSMTETMP 54,43,23,14.5 37 KMMHNANMSMTE 6.8 38 KMMHNANMSMTE 6 39 NA 54.16.5 40 AMPCBNASMTE 38 41 AMPCBCMNMSMTE 23 42 NASMXSMTETOBTMP 22 43 CMMHNASMXSMTETMP 38 44 CMKMNANMSMXSMTETMP 45,33 45 AMPCBCMKMNANMSMXSMTETOBTMP 58,48 46 AMPCBMHSMTE 48,38 47 AMPCBMHSMTE 51,38 Percentage of E. coli isolates No of antibiotics resistnat to antibiotics 1 6.3 2 2.1 3 6.3 4 19 5 14.8 6 14.8 7 14.8 8 2.1 9 4.2 10 4.2 11 8.5 12 2.1 13 0 14 0 15 0 Figure 1. Percentage of Escherichia coli isolates resistant to multiple antibiotics. Note: Table made from bar graph. Percentage of resistance Antibiotics of E. coli isolates AK 0 AMP 36.8 CB 36.8 CTX 0 CM 31.6 GM 10.5 KM 56.1 MH 33.3 NA 80.7 NM 52.6 SMX 42.1 SM 66.7 TE 94.7 TOB 14 TMP 43.9 Figure 2. Percentage of Escherichia coli isolates resistant to each of the antibiotics tested. Note: Table made from bar graph.