Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 © 1999 Blackwell Science Ltd 278 Antibiotic resistance of Escherichia coli isolated from poultry workers, patients and chicken in the eastern province of Saudi Arabia Mastour S. Al-Ghamdi 1 , Fikry El-Morsy 2 , Zaki H. Al-Mustafa 1 , Mustafa Al-Ramadhan 3 and Mohammad Hanif 4 1 Department of Pharmacology, College of Medicine, King Faisal University 2 Department of Microbiology, College of Medicine, King Faisal University 3 College of Veterinary Medicine, King Faisal University 4 Department of Family and Community Medicine, College of Medicine, King Faisal University, Saudi Arabia Summary The prevalence of antibiotic-resistant Escherichia coli isolates from faecal samples from 117 poultry industry workers, 100 patients and119 healthy chicken were compared. Resistance of E. coli chicken isolates to ampi- cillin, chloramphenicol, gentamicin, spectinomycin, tetracycline and trimethoprim ϩ sulphamethoxazole (TMP ϩ SMX) (range 57% – 99.1%) were significantly higher than those isolated from patients (range 21.9% – 71.4%) and workers (range 35% – 71.8%). However, for drugs not used in poultry, such as amoxi- cillin ϩ cluvalanate (AMX ϩ CLV), ceftazidime and nitrofurantoin, resistance rates of chicken isolates (range 0% – 2.6%) were significantly lower than those of patient isolates (range 8.7% – 30%). Resistance to spectinomycin reached 96% in E. coli chicken isolates and 71% in organisms isolated from humans. Use of this drug in Saudi Arabia is mostly limited to veterinary purposes. Multidrug resistance is alarmingly high in all groups but was highest in chicken isolates (77.4%). Serotyping of E. coli isolates showed that 27% of the organisms isolated from patients were overlapping with 10.9% of the chicken isolates, indicating the possi- bility of chicken being a source of the resistance pool for humans. We therefore call for the banning of antibiotics in the poultry industry as growth promoters and recommend that their use be restricted to treat- ing infections. keywords Escherichia coli, microbial, antibiotics, antibiotic resistance, poultry, Saudi Arabia. correspondence Dr Mastour Al-Ghamdi, Department of Pharmacology, College of Medicine, King Faisal University, PO Box 2114, Dammam 31451, Saudi Arabia Introduction Antibiotics are widely used in poultry production as growth promoters or to control infectious disease. This practice is reported to have caused high resistance to antimicrobial agents in normal chicken flora (Swan Report 1969; Smith 1974; Aronson 1975; Linton 1977; Allan et al. 1993) and path- ogenic organisms (Amara et al. 1995). These resistant micro- organisms may act as a possible source for the transfer of antimicrobial resistance to human pathogens (Bebora et al. 1994). Plasmid and transpon-mediated resistance is widely transmitted between different bacterial species and genera including human pathogens (Wise et al. 1985; Davies 1994). Multidrug-resistant strains of E. coli are prevalent in both human and animal isolates in different parts of the world (Mahipal et al. 1992; Bebora et al. 1994; Amara et al. 1995). E. coli is a common normal flora organism in the gastro- intestinal tract of animals and man (Jawetz et al. 1984) but may become pathogenic to both (Jacobs et al. 1970; Marier et al. 1973; Burkhanova 1980; Levine 1987). Serious outbreaks of gastrointestinal illness caused by foodborne pathogenic E. coli, especially 0157:H7, have occurred during the past two decades (Armstrong et al. 1996). Thus, resistant strains of E. coli arising from the exposure of animals to antimicrobials may possibly become infectious organisms in humans. Antimicrobial agents are widely used in the poultry indus- try of Saudi Arabia. In fact, our own survey showed that 28 antimicrobial agents were available for poultry use in the Eastern Province of Saudi Arabia. These include ␤– lactams, tetracyclines, aminoglycosides, macrolides, fluoroquinolones, TMIH392 Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 M. S. Al-Ghamdi et al. E. Coli antibiotic resistance in Saudi Arabia © 1999 Blackwell Science Ltd 279 linconasomides and sulphonamides. They are mainly added, often concomitantly, to drinking water for infection prophy- laxis. Twenty of these antimicrobial agents are also com- monly used in the treatment of human infections. However, no study was so far conducted in Saudi Arabia to evaluate resistance to antibiotics in poultry and its possible impact on human health. Our aims were to investigate and compare the patterns of resistance of E. coli isolated from poultry industry workers, patients and chicken, and to identify the common serotypes of E. coli isolated from humans and poultry. Materials and methods The susceptibility patterns of 119, 117 and 100 E. coli organ- isms isolated from live healthy chicken in local slaughter shops, poultry industry workers and patients in King Fahd Hospital of the University, Al-Khobar, Saudi Arabia, respect- ively, were determined. Specimens were directly inoculated on MacConkey agar, eosin methylene blue and xylose lysine des- oxycholate agar media and growth was identified by standard laboratory methods including Gram stain and API system (BioMerieux SA, Marcy Etoile, France). The Bauer-Kirby disc diffusion method (Bauer et al. 1966) was used to test suscepti- bility of the isolated organisms to ampicillin (10 ␮g), amoxi- cillin ϩ clavulanic acid (AMX ϩ CLV) (Augmentin, 30 ␮g), ceftazidime (75 ␮g), chloramphenicol (30 ␮g), tetracycline (30 ␮g), gentamicin (10 ␮g), spectinomycin (10 ␮g), tri- methoprim ϩ sulphamethoxazole (TMP ϩ SMX) (Septrin, 25 ␮g) and nitrofurantoin (100 ␮g). All antibiotic discs were obtained from Oxoid (Unipath Ltd, Basingstoke, UK). Interpretation followed criteria recommended in the National Committee for Clinical Laboratory Standards (NCCLS 1993). In addition, E. coli organisms isolated from chicken and patients were serotyped using E. coli antisera (Denken Seiken, Tokyo, Japan). All data were recorded and analysed using SPSS/PC soft- ware. Results were statistically analysed using the Spearman correlation coefficient test and Student’s t-test, with a signifi- cance level of P Ͻ 0.05. Results The susceptibility of E. coli isolates from chicken and patients to the tested antibiotics is summarised in Table 1. The highest resistance rate observed in the chicken E. coli isolates was to tetracycline (99.1%) followed by those to spectinomycin (95.7%), TMP ϩ SMX (92.2%), gentamicin (89.7%), ampicillin (88.7%) and chloramphenicol (57.0%). AMX ϩ CLV, ceftazidime and nitrofurantoin remained highly effective with resistance rates Յ 2.6%. The highest rate of resistance in E. coli isolated from patients was to spectinomycin (71.4%), followed by ampicillin (70.7%), tetra- cycline (64.7%), TMP ϩ SMX (46.3%) and AMX ϩ CLV (30.0%). Moderate or low resistance rates were noted with cef- tazidime, nitrofurantoin and gentamicin. Both groups showed similar patterns of resistance to the tested antibiotics, with a Spearman correlation coefficient of 0.7448 (P ϭ 0.021). However, resistance rates to individual drugs in E. coli iso- lated from patients remained significantly lower than those of E. coli chicken isolates with the exception of resistance to cef- tazidime, AMX and nitrofurantoin (Table 1). The Spearman test also revealed a highly significant corre- lation (coefficient ϭ 0.9289, P ϭ 0.000) between the resist- ance pattern of E. coli isolated from poultry industry workers and E. coli isolated from chicken (Table 2). However, despite the similarity of resistance patterns to the tested antibiotics in E. coli-chicken E. coli-patient isolates isolates t-test No. % No. % Drugs tested resistance tested resistance P-value Significance Ampicillin 115 88.7 99 70.7 0.0002 S AMXϩCLV 116 02.60 99 30.0 0.0002 S Ceftazidime 114 00.0 92 08.70 – NA Chloramphenicol 113 57.0 92 28.3 0.0001 S Tetracycline 116 99.1 99 64.7 0.0000 S Gentamicin 116 89.7 96 21.9 0.0001 S Spectinomycin 116 95.7 91 71.4 0.0000 S TMPϩSMX 116 92.2 95 46.3 0.0000 S Nitrofurantoin 116 02.6 74 10.8 0.0456 S S, Significant; NA, Not applicable. Spearman Correlation coefficient ϭ 0.7448, P ϭ 0.021 Table 1 Comparison of pattern of resistance of E. coli isolates from chicken and patients Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 M. S. Al-Ghamdi et al. E. Coli antibiotic resistance in Saudi Arabia © 1999 Blackwell Science Ltd 280 both groups, resistance rates remained significantly lower in E. coli isolated from the workers than those from chicken. Multidrug resistance rates of E. coli organisms isolated from chicken were significantly greater than those of patient isolates (Table 3). This became more evident when comparing the rates of resistance to four drugs or more in both groups (78.3% and 77.4% against 8.8% and 7.7%, respectively). In addition, 20.5% of E. coli organisms isolated from workers were multiply resistant to the same drugs, which was also sig- nificantly (P ϭ 0.0124) more than in patients’ isolates. Serotyping of 119 and 100 E. coli organisms isolated from chicken and patients, respectively, is presented in Table 4. Only 30.3% of chicken E. coli isolates and 38% of patient isolates were serotypeable. Twenty-one serotypes were found among E. coli isolated from chicken, most commonly 0114 (6 isolates), 06 (4 isolates) and 01 (3 isolates). There were 15 serotypes in human E. coli isolates with 06 (14 isolates), 027 (6 isolates) and 018 (3 isolates) being the most common ones. Seven serotypes (01, 06, 08, 015, 027, 0119 and 0167) were found in both groups. These represent 10.9% of the poultry E. coli isolates and 27% of the E. coli isolated from patients (Table 4). Discussion We investigated the resistance of E. coli isolated from poultry industry workers and patients to 9 antimicrobial agents commonly used in the poultry industry and/or for patients in E. coli-chicken E. coli-patient isolates isolates t-test ————————— ————————— —————————– No. % No. % Drugs tested resistance tested resistance P-value Significance Ampicillin 115 88.7 117 53.8 0.0000 S AMXϩCLV 116 02.60 117 05.1 0.4472 NS Ceftazidime 114 00.0 117 00.0 – NA Chloramphenicol 113 57.0 117 35.0 0.0006 S Tetracycline 116 99.1 117 58.1 0.0000 S Gentamicin 116 89.7 117 37.6 0.0000 S Spectinomycin 116 95.7 117 71.8 0.0000 S TMPϩSMX 116 92.2 117 53.0 0.0000 S Nitrofurantoin 116 02.6 117 06.0 0.2892 NS S, Significant; NA, Not applicable; NS, Not significant. Spearman Correlation coefficient ϭ 0.9289, P ϭ 0.000 E. coli-chicken E. coli-patient t-test ———————– ———————– ———————–—– No. % No. % Drugs tested resistance tested resistance P-value Significance Ampicillin, chloramphenicol 113 49.6 92 20.6 0.0000 S Ampicillin, gentamicin 115 82.6 96 16.7 0.0000 S Ampicillin, spectinomycin 115 88.7 91 48.4 0.0000 S Ampicillin, tetracycline 115 89.6 99 55.6 0.0000 S Ampicillin, TMPϩSMX 115 85.2 94 43.6 0.0000 S Ampicillin, gentamicin, 115 78.3 91 08.8 0.0000 S tetracycline & TMPϩSMX Ampicillin, gentamicin, 115 77.4 91 07.7 0.0000 S spectinomycin, tetracycline & TMPϩSMX S, Significant Table 2 Comparison of pattern of resistance of E. coli isolates from chicken and laborers Table 3 Comparison of multi-resistance of E. coli-chicken isolates and E. coli- patients isolates Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 M. S. Al-Ghamdi et al. E. Coli antibiotic resistance in Saudi Arabia Van-den-Bogaard and Stobberingh (1996) recently called for banning the use of antibiotics as growth promoters in ani- mals in the Netherlands. Spectinomycin is an aminocyclitol compound related struc- turally to aminoglycosides and used by veterinarians as a broad spectrum antibiotic against gram negative bacteria including E. coli, Klebsiella, Salmonella, Proteus and Enterobacter organisms (Allen et al. 1993). E. coli organisms isolated from chicken were almost totally resistant to tetra- cycline and spectinomycin. This might be explained by the fact that both are heavily used in the poultry industry in Saudi Arabia and could be the result of misuse or antagonism effect between the two drugs, especially when used concur- rently (Allen et al. 1993). High resistance rates were also noted with TMP ϩ SMX and chloramphenicol in E. coli iso- lated from poultry despite the fact that the latter is not licensed for veterinary use in Saudi Arabia. This again could be associated with the misuse of spectinomycin as reported by Ginns et al. (1996). Although the high resistance rate of E. coli chicken isolates to spectinomycin may be attributed to the misuse of this drug in the poultry industry, the reasons for high resistance in human E. coli isolates are not fully understood, as its clinical use is limited to second-line therapy for Neisseria gonorrhea (Kapusnik-Uner et al. 1996). However, such resistance could be either natural or due to poultry as a possible source of the spectinomycin resistance pool for humans. Exposure to antibiotics at work may also contribute to increased resistance rates. The Spearman correlation co- efficient test revealed a significant correlation between the pattern of resistance to antibiotics in E. coli organisms from chicken and from patients (Table 1) as well as from workers (Table 2). These findings not only demonstrate the similarity of the organisms’ attitude toward the investigated antibiotics, but also confirm the effect of work exposure to antibiotics. This is corroborated by multiple resistance to four or five antimicrobial agents commonly used in the poultry industry. Bongers et al. (1995) noted a significantly higher level of resistance to oxytetracycline and ampicillin in poultry indus- try workers than in pig husbandry workers or those not working with animals. This was attributed to the differences in exposure to antibiotics at work. The E. coli isolated from chicken belonged to 21 serotypes, with 69.6% being nontypeable. The most frequent serotypes in this study were 0114, 06 and 01. Similarly, Ngeleka et al. (1996) reported that only 38.4% of 39 E. coli isolated from broiler chickens with cellulitis were typeable, but the most frequent serotypes in their study were 025 and 078. In con- trast to our findings, Allan et al. (1993) found that 61% of 44 avian E. coli isolates were typeable, with 01, 02 and 078 being the most frequent serotypes. E. coli isolated from patients belonged to 15 serotypes, © 1999 Blackwell Science Ltd 281 the Eastern Province of Saudi Arabia. E. coli isolated from chicken clearly demonstrated high resistance rates to all tested antibiotics commonly used in the poultry industry but not to those which are mainly used for patients (AMX ϩ CLV, ceftazidime and nitrofurantoin) (Table 1). By contrast, higher resistance rates to these drugs were noted in organisms isolated from human patients. This suggests that the extent of resistance to an antibiotic is associated with the extent of its use. The high antibiotic resistance rate of organ- isms isolated from animals is not a phenomenon unique to Saudi Arabia. A recent report by Blanco et al. (1997) in Spain showed that up to 67% of avian E. coli strains were resistant to TMP ϩ SMX and that resistance to the new fluoro- quinolones was also increasing. Similarly, Son and Gulam (1995) in Malaysia isolated plasmids of E. coli with high resistance rates to 9 antibiotics in clinical use for humans. Table 4 Serotypes of organisms isolated from chicken and patients Serotyping E. coli-chicken E. coli-patient isolates isolates Polyvalent Serotype non-serotypeable 83 62 1 0001 03 02 1 0119 01 01 1 0086 02– 1 0111 01– 1 0128 01– 2 0146 02– 2 0152 – 01 2 0166 01– 3 0018 – 03 3 0114 06– 4 0006 0414 4 0027 01 06 4 0148 01– 4 0159 01– 4 0048 01– 4 0029 – 01 4 0078 – 02 5 0053 01– 5 0063 02– 5 0167 02 01 6 0008 01 01 6 0015 01 02 6 0028 – 01 6 0169 01– 7 0112 – 01 7 0144 – 01 8 0029 – 01 8 0143 01– 8 0152 02– Total serotyped 36 (30.3%) 38 (38%) Overlapped serotype 13 (10.9%) 27 (27%) Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 M. S. Al-Ghamdi et al. E. Coli antibiotic resistance in Saudi Arabia with 62% being nontypeable (Table 4). However, serotypes of 27% of E. coli organisms isolated from human patients were also found in chicken isolates, with 06 and 027 being the most common serotypes in both groups (Table 4). These results suggest that chicken may act as a possible source of human pathogenic organisms. Cherry et al. (1961) reported that some serotypes, such as 055, 0126, 086, 026 and 0119, which were isolated from chicken, were capable of producing infec- tion in children. More recently, E. coli 0157:H7 has been rec- ognized as a dangerous foodborne pathogen (Griffin 1995; Armstrong et al. 1996). In conclusion, our data demonstrate alarmingly high individual and multiple resistance to antibiotics in E. coli, reflecting the misuse of these agents in the poultry industry. Since chicken may act as a possible source of pathogenic organisms in humans, we concur with the calls to ban the use of antibiotics as growth promoters and recommend the re- striction of their use to treat infections in the poultry industry. Acknowledgements This work was funded by the King Abdulaziz City of Science and Technology (Grant no. AT-15–79) to whom we express our gratitude. Our sincere thanks also go to Dr Abdul- Rahman Qurashi and the technical staff at the Microbiology Laboratories at King Fahd Hospital of the University and the College of Medicine for their assistance. We also thank Prof A. Ghandour, Department of Microbiology, King Faisal University, for reviewing the manuscript. References Allan BJ, Van-den-Hurk W & Potter AA (1993) Characterization of Escherichia coli isolated from cases of avian colibacillosis. Canadian Journal of Veterinary Research 57, 146–151. Allen DG, Pringle X, Smith D, Conlon PD & Burgmann PM (1993) Handbook of Veterinary Drugs. Lippincott, Philadelphia, pp. 257–258. Amara A, Ziani Z & Bouzoubaa K (1995) Antibioresistance of Escherichia coli strains isolated in Morocco from chickens with colibacillosis. Veterinary Microbiology 43, 325–330. Armstrong GL, Hollingsworth J & Morris JG Jr (1996) Emerging foodborne pathogens: Escherichia coli 0157: H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiological Review 18, 29–51. Aronson AL (1975) Potential impact of the use of antimicrobial drugs in farm animals on public health. Presented at the meeting on pharmacology in the animal health sector. Sept. 23–24, 1975. Colorado State University, Fort Collins. Bauer AW, Kirby WMM, Sherris JC & Turk M (1966) Antibiotic sus- ceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45, 493–496. Bebora LC, Oundo JO & Yamamoto H (1994) Resistance of E. coli strains, recovered from chickens, to antibiotics with particular ref- erence to trimethoprim-sulfamethoxazole (Septrin). East African Medical Journal 71, 624–627. Blanco JE, Blanco M, Mora A & Blanco J (1997) Prevalence of bac- terial resistance to quinolones and other antimicrobials among avian Escherichia coli strains isolated from septicemic and healthy chickens in Spain. Journal of Clinical Microbiology 35, 2184–2185. Bongers JH, Franssen F, Elbers AR & Tielen MJ (1995) Antimicrobial resistance of Escherichia coli isolates from the faecal flora of veterinarians with different professional specialties. Veterinarians’ Quarterly 17, 146–149. Burkhanova KHK (1980) Properties of E. coli strains isolated fron diseased fowls. Veterinarria Moscow USSR 10, 66. Cherry WB, Thomason BM, Pomales Lebron A & Ewing WB (1951) Rapid presumptive identification of entero pathogenic Escherichia coli in faecal smears by means of fluorescent antibody. 3. Field eval- uation. Bulletin of the of the World Health Organization 25, 159–171. Davies J (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264, 375–382. Ginns CA, Browning GF, Benham ML, Anderson GA & Whithear KG (1996) Antimicrobial resistance and epidemiology of Escherichia coli in broiler breeder chickens. Avian Pathology 3, 591–605. Griffin PM (1995) Escherichia coli 0157: H7 and other enterohemor- rhagic Escherichia coli. In: Infections of the Gastrointestinal Tract (ed. M.J. Blaser et al.) Raven Press, New York, pp. 739–761. Jacobs SI, Halizel A & Wolman B (1970) Outbreak of infantile gastroenteritis caused by E. coli 0114. Archives of Disease in Childhood 45, 243. Jawetz E, Melnick J & Adelberg EA (1984) Review of Medical Microbiology, 16th edn. Long Medical Publication, Los Altos. Kapusnik-Uner JE, Sande MA & Chambers HF (1996) Tetracyclines, chloramphenicol, erythromycin and miscellaneous antibacterial agents. In: The Pharmacological Basis of Therapeutics 9th edn. (ed. J.G. Hardman et al.) McGraw Hill, New York, p. 1143. Levine M (1987) Escherichia coli that cause diarrhea: enterotoxi- genic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent. Journal of Infectious Diseases 155, 377–390. Linton AH (1977) Antibiotics, animals and man. An appraisal of a continuous subject. In: Antibiotics and Antibiosis in Agriculture (ed. M Woobine), Butterworths, London, pp. 315–343. Mahipal S, Chaudhry MA, Yadava JNS & Sanyal SC (1992) The spectrum of antibiotic resistance in human and veterinary isolates of Escherichia coli collected from 1984 to 1986 in Northern India. Journal of Antimicrobial Chemotherapy 29, 159–168. Marier R, Wells JG, Swanson RC, Collahan W & Mehlaman IJ (1973) An outbreak of enteropathogenic E. coli foodborne disease traced to imported French cheese. Lancet ii, 1376. National Committee for Clinical Laboratory Standards (NCCLS) (1993) Performance Standards for Antimicrobial Disk Susceptibility Tests Approved Standard M2 -A5. 5th edn. NCCLS, Villanova, Pa. Ngeleka M, Kwaga JK, White DG, Whittam TS, Riddell C & Goodhope R (1996) Escherichia coli cellulitis in broiler chickens: clonal relationships among strains and analysis of virulence-asso- ciated factors of isolates from diseased birds. Infection and Immunity 64, 3118–3126. © 1999 Blackwell Science Ltd 282 Tropical Medicine and International Health volume 4 no 4 pp 278–283 april 1999 M. S. Al-Ghamdi et al. E. Coli antibiotic resistance in Saudi Arabia 4190), HMSO, London. Van-den-Bogaard AE & Stobberingh EE (1996) Time to ban all antibiotics as animal growth promoting agents? Lancet 348, 619. Wise RJ, Towner KJ, Webster CA, Slack RC & Jones TO (1985) Trimethoprim resistance plasmids in Escherichia coli isolated from diarrhoeae in cattle, pigs and sheep. Journal of Applied Bacteriology 58, 555–561. © 1999 Blackwell Science Ltd 283 Smith HW (1974) Veterinary and food aspects of drug resistance. Journal of Science in Food and Agriculture 25, 227–237. Son RI & Gulam RRA (1995) Plasmids and antimicrobial susceptibil- ity of Escherichia coli with special reference to poultry isolates in Sarawak. Journal of Veterinar Malaysia 7, 21–25. Swan Report (1969) Report of the Joint Committee on the use of antibiotics in animal husbandry and veterinary medicine. (Cmnd . patterns of resistance of E. coli isolated from poultry industry workers, patients and chicken, and to identify the common serotypes of E. coli isolated. 27% of the organisms isolated from patients were overlapping with 10.9% of the chicken isolates, indicating the possi- bility of chicken being a source of