Isolation and Antibiotic Susceptibility of Salmonella, Shigella, and Campylobacter from Acute Enteric Infections in Egypt

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1 J Isolation HEALTH and POPUL antibiotic NUTR susceptibility 2000 Jun;18(1):33-38 of Salmonella, Shigella, 2000 and ICDDR,B: Campylobacter Centre in for Egypt Health and Population Research 33 ISSN $ Isolation and Antibiotic Susceptibility of Salmonella, Shigella, and Campylobacter from Acute Enteric Infections in Egypt Momtaz O. Wasfy 1, Buhari A. Oyofo 1, John C. David 1, Tharwat F. Ismail 1, Atef M. El-Gendy 1, Zaynab S. Mohran 1, Yehia Sultan 2, and Leonard F. Peruski, Jr. 1* 1 U.S. Naval Medical Research Unit No. 3, Cairo; and 2 Abbassia Fever Hospital, Cairo, Egypt Present address: US NAMRU No. 2, Jakarta, Indonesia ABSTRACT While Campylobacter, Salmonella, and Shigella remain major contributors to acute enteric infections, few studies on these pathogens have been conducted in Egypt. From January 1986 to December 1993, 869 Salmonella, Shigella and Campylobacter strains were isolated from stool specimens from 6,278 patients, presenting to the Abbassia Fever Hospital, Cairo, Egypt, with acute enteric infections. Salmonella predominated, totalling 465 isolates, followed by Shigella with 258 isolates, and Campylobacter with 146 isolates. Of the Shigella isolates, 124 were Shigella flexneri, 49 were S. sonnei, 47 were S. dysenteriae (mainly serotype 1, 2, and 3), and 38 were S. boydii. Campylobacter spp. comprised 92 Campylobacter jejuni and 54 C. coli isolates. Isolation of Salmonella was highest during the months of February-March, June-July, and October- November, while that of Shigella was maximal from July to October. Isolation of Campylobacter increased during May-June and again during August-October. Although Salmonella was sensitive to amikacin, aztreonam, ceftriaxone, and nalidixic acid, it was, however, resistant to erythromycin, streptomycin, ampicillin, chloramphenicol, and tetracycline. Shigella (>80%) was sensitive to amikacin, ceftriaxone, cephalothin, sulphamethoxazole-trimethoprim (except S. sonnei), aztreonam, and nalidixic acid. Resistance (>50%) was noted only for ampicillin, chloramphenicol, and tetracycline. C. jejuni and C. coli were resistant to cephalothin, aztreonam, and streptomycin. Some of the above antibiotics were employed to characterize the Egyptian isolates, but did not have any clinical utility in the treatment of diarrhoea. Significant differences (p<0.05) were observed in the resistance profiles of Shigella and Salmonella between late 1980s and early 1990s. The results suggest the use of fluoroquinolones or a third-generation cephalosporin as an empirical treatment of enteric diseases. However, alternative control strategies, including the aggressive development of broadly protective vaccines, may be more effective approaches to curbing morbidity and mortality due to acute enteric infections. Key words: Salmonella; Shigella; Campylobacter; Antibiotic resistance; Diarrhoea; Drug resistance, Microbial; Microbial sensitivity tests; Dysentery, Bacillary; Salmonella infections Correspondence and reprint requests should be addressed to: Commanding Officer (Attn: Code 101F) US Naval Medical Research Unit No. 3 PSC 452, Box 5000 FPO AE USA Fax: + (011-20) boushrah@namru3.navy.mil INTRODUCTION Diarrhoeal disease and enteric infections are major causes of morbidity and mortality in the developing world, resulting in over a quarter of all childhood deaths (1). Globally, Salmonella and Shigella remain major contributors to acute enteric infections (2,3) with nontyphoidal Salmonella isolated in increasing numbers in diverse geographic regions (3,4). Campylobacter has

2 34 J HEALTH POPUL NUTR Jun 2000 Wasfy MO et al. emerged as a significant cause of gastroenteritis (5). When clinical laboratories include a screen for Campylobacter in routine enteric culture procedures, the percent-recovery for this organism often exceeds that of Salmonella and Shigella (6). However, screening for Campylobacter in acute enteric infections is often not a routine matter due to its relatively recent link to human disease and the complexity of procedures for its isolation and identification (7,8). As a result, there is little information available specifically on Campylobacter in acute enteric infections or antimicrobial resistance from the developing world (8). In 1984, the Egyptian Ministry of Health estimated infant mortality as 72 cases per 1,000 (7.2%), of which 43 cases (4.3%) were due to acute enteric infections (9). In a more recent report (10), mortality decreased in the early 1990s to 34 cases per 1,000, of which 9 cases were due to diarrhoea. Descriptive characterization of bacterial pathogens involved, including antibiotic susceptibility, remains poor, as few such studies have been conducted in Egypt on the causative bacterial agents and their antibiotic resistance profiles in the indigenous population (9,11). Because of these limitations in the available regional data, we analyzed the results of an ongoing hospitalbased study to evaluate the percent-recovery, seasonality, and antibiotic resistance of Campylobacter, Shigella, and Salmonella isolated from patients with acute enteric infections reported to a major regional hospital in Egypt from 1986 to Sample collection METHODS AND MATERIALS Single stool samples from each of 6,278 patients with acute enteric infections were examined for the presence of Salmonella, Shigella, and Campylobacter over the course of this study from 1986 to All patients were Egyptian nationals with a mean age of 14.1 years (ranging from 4.8 to 23.4 years) admitted to the Abbassia Fever Hospital, Cairo, Egypt, based on clinical signs of acute diarrhoea or enteric fever. This hospital is one of two major fever hospitals in the greater Cairo metropolitan area which provide low-cost medical care to a regional population in excess of 15 million. Stool specimens were transferred within one hour of their collection from patients to the adjacent bacteriology laboratory at the U.S. Naval Medical Research Unit No. 3 for analysis. Bacterial analysis Stool samples were streaked onto selective media (Difco Laboratories, Detroit, Michigan, USA) for the isolation and identification of Campylobacter, Salmonella, and Shigella. Briefly, stools were streaked onto MacConkey, Salmonella-Shigella (SS), Xylose-Lysine-Deoxycholate (XLD), Hektoen-Enteric agars (HE), and were incubated at o C for primary isolation. Inoculation into Selenite-F enrichment broth and subculturing on SS agar were performed to improve the recovery of Salmonella (12). Suspect isolates were identified, using the API- 20E rapid identification kits (Bio-Merieux Vitek Inc, Hazelwood, Missouri, USA) and commercial antisera (Difco Laboratories) as appropriate. For the isolation of Campylobacter, stools were streaked onto modified Skirrow s agar and incubated at 42 o C in an icroaerophilic atmosphere. Suspect colonies were identified as Campylobacter isolates on the basis of morphology, Gram stain, motility, as well as oxidase and catalase tests. C. jejuni and C. coli (vs C. lari) were differentiated on the basis of hippurate and indoxyl acetate hydrolysis. Typically, C. jejuni is positive for the two tests, while C. coli is positive for indoxyl acetate hydrolysis only (12). Antibiograms for Salmonella and Shigella isolates were determined by the disk-diffusion method (13) on Mueller-Hinton agar, using 12 antibiotics: amikacin (30 µg), ampicillin (10 µg), aztreonam (30 µg), cephalothin (30 µg), chloramphenicol (30 µg), ceftriaxone (30 µg), erythromycin (15 µg), gentamicin (10 µg), nalidixic acid (30 µg), streptomycin (10 µg), tetracycline (30 µg), and sulphamethoxazole-trimethoprim (25 µg). Antibiograms for Campylobacter were also determined by the diskdiffusion method, except that 5% sheep blood was added to the Mueller-Hinton agar and susceptibility to sulphamethoxazole-trimethoprim was not determined because of the potential for erroneous results on bloodbased media. RESULTS In total, 869 Salmonella, Shigella, and Campylobacter strains were isolated from single stool specimens collected from each of 6,278 patients diagnosed as having acute enteric infections (Table 1). Culturepositive specimens constituted 13.9% of the total, with the identification of 869 pathogens. Salmonella predominated, totalling 465 isolates (54%), followed by Shigella with 258 isolates (30%), and Campylobacter with 146 isolates (17%). Of the 465 Salmonella isolates, S. typhi comprised the majority with 245 isolates (53%). Salmonella group B had 49 isolates (10%), group D 41 isolates (9%), group C2 40 isolates (9%), and group C1 36 isolates (8%). Of the 258 Shigella isolates, S. flexneri was most frequently identified, comprising 124 isolates (48%), followed by S. sonnei with 49 isolates (19%), S. dysenteriae with 47 isolates (18%, mainly serotype 1, 2, and 3), and S. boydii with 38 isolates (15%). All 146 Campylobacter strains were identified as either C. jejuni (92 isolates, 63%) or C. coli (54 isolates, 37%). The figure shows the percent-recovery of each of the three genera on a monthly basis. Salmonella isolation was highest in the month of February-March, June-July, and October-November, and was lowest in April, September, and December. Isolation of Shigella was generally lower during November-March and maximal from July to October. Campylobacter isolation was more uniform over the course of the year, but increased during

3 Isolation and antibiotic susceptibility of Salmonella, Shigella, and Campylobacter in Egypt 35 May-June and again during August-October, which roughly corresponded to the periods of the lowest recovery of Salmonella. Antibiotic resistance for those isolates available for testing is summarized in Table 2. Most Salmonella Table 1. Enteric pathogens identified during Species/serogroup Total no. of isolates Cumulative rate a Percentage S. typhi Salmonella Gr. B Salmonella Gr. D Salmonella Gr. C Salmonella Gr. C Salmonella species Salmonella Gr. E S. paratyphi A Total Salmonella S. flexneri S. sonnei S. dysenteriae b S. boydii Total Shigella C. jejuni C. coli Total Campylobacter Total a Based on the number of specimens that were culture-positive b Mainly serotype 1, 2, and 3 Month Salmonella; Shigella; Campylobacter Mean number of positive patients per month 8.6±3.0 (range= ) Mean number of total patients cultured per month 66.5±11.9 (range= ) Fig. Percentage of culture-positive faecal specimens by pathogen ( ) isolates, irrespective of species, were sensitive to amikacin, aztreonam, ceftriaxone, and nalidixic acid. In contrast, more than 70% of all isolates were resistant to streptomycin, erythromycin, ampicillin, chloramphenicol, sulphamethoxazole-trimethoprim, and tetracycline. The four Shigella species shared similar susceptibility profiles for most antibiotics. They were resistant to ampicillin (except S. sonnei), cephalosporins, chloramphenicol (except S. boydii and S. sonnei), streptomycin, and erythromycin. Susceptibility profiles of both C. jejuni and C. coli were similar during the study period (Table 2). They were resistant to cephalothin, aztreonam, streptomycin, and ceftriaxone (>56%). Less than 10% were resistant to amikacin, chloramphenicol, erythromycin, and gentamicin. C. coli was more resistant than C. jejuni to both ampicillin (69% vs 48%) and tetracycline (24% vs 6%), while C. jejuni was more resistant than C. coli to nalidixic acid (40% vs 24%). When susceptibility of all the isolates was compared between late 1980s and early 1990s, a significant increase (p<0.05) in the resistance profiles of some Salmonellae to chloramphenicol and cephalothin was observed (Table 3). Similarly, in recent years, resistance of Shigella to sulphamethoxazole-trimethoprim had significantly increased, but that of tetracycline had decreased (p<0.05). DISCUSSION Hospital patients are generally assumed to reflect the relative importance and seriousness of prevalent illnesses within a region (14). Our investigation took advantage of the large patient population suffering from acute enteric diseases admitted to the Abbassia Fever Hospital in Cairo to determine the percent isolation and antibiotic susceptibility of Salmonella, Shigella, and Campylobacter over an 8- year period from 1986 to The predominant bacterial pathogen isolated was Salmonella, followed by Shigella and Campylobacter, indicating that they remain significant threats to health in this region. Apart from S. typhi, which was the most common pathogen in this study, isolates of other Salmonella serogroups were found in approximately 1% or less of specimens. Other reports from neighbouring countries found total Salmonella isolation ranging from 2% to 18% of specimens (15-18). We found a greater percentage of specimens positive for S. typhi or S. paratyphi. This could reflect the source of our faecal specimens that were obtained from a hospital ward that receives a large

4 36 J HEALTH POPUL NUTR Jun 2000 Wasfy MO et al. Table 2. Percent antibiotic resistance by pathogen Salmonella Shigella Campylobacter S. dysen. S. flex. S. boyd. S. sonn. C. coli C. jejuni Species Gr. B Gr. C1 Gr. C2 Gr. D Gr. E S. pararararara. Antibiotic (33) a (110) (24) (24) (27) (65) (13) S t phiphiphiphiphi (41) (28) (31) (33) (13) (6) (235) Amikacin Ampicillin Aztreonam Cephalothin Ceftriaxone Chloramphenicol Erythromycin Gentamicin Nalidixic acid Streptomycin ND 78 SxT b ND ND Tetracycline a The value in parenthesis is the number of isolates tested b Sulphamethoxazole-trimethoprim number of typhoid fever cases annually. The disparity in isolation between typhoidal and non-typhoidal serogroups may be due to increased exposure or higher recurrent susceptibility as a result of the indiscriminate use of antibiotics in the general population. Our findings support prior observations that antibiotic resistance of S. typhi and S. paratyphi varies with the locality and prevailing treatment regimens, likely through the acquisition of resistance plasmids (2,19,20). It should be recalled, however, that some antibiotics, such as amikacin, aztreonam, cephalothin, erythromycin, gentamicin, and streptomycin, tested in this study, have no clinical utility in the treatment of diarrhoea, but may help characterize the isolates. In developing countries, S. flexneri is the most common isolate from acute enteric diseases, while S. sonnei is more prevalent in the developed world (21, 22). Our results are in good agreement with this trend. The distribution and seasonality of isolation are similar to that reported in the neighbouring countries of Saudi Arabia, Jordan, Lebanon, Kuwait, and Israel (8,16-18, 23), with the exception of one study from Saudi Arabia which reported over 17% of all specimens as positive for Shigella (15). The antibiotic resistance pattern of Shigella isolates was similar to that of Salmonella isolates, and is supported by studies from the neighbouring countries and the developed world (22,24-27). Regional studies have also reported similar findings with S. flexneri isolates, being more resistant to ampicillin, chloramphenicol, and gentamicin than S. sonnei isolates (16-18,22). Although Campylobacter was isolated from the Egyptian patients throughout the year, two peak intervals were observed--one from May to June and another from September to October. An earlier study (11) reported an increased rate of occurrence of Campylobacterassociated diarrhoea during the moderate or rainy seasons in Egypt. Other studies have suggested that the seasonal trends for Campylobacter infection may be less evident in the tropical and subtropical countries (5,6). The annual percent-isolation of Campylobacter averaged 2.3%, identical to that reported from Kuwait (18), half that reported in similar studies in Jordan and Saudi Arabia (8,17), but over twice that reported in Israel (28). In agreement with previous reports (6,11), C. coli was recovered from patients with acute infections, despite the fact that the pathogenicity of this species has not been fully determined in human volunteer studies. Both C. coli and C. jejuni had similar levels of antibiotic resistance which is in agreement with a previous report (29). The emergence of resistance to quinolones in Campylobacter may be an ominous trend that has not been previously noted in Egypt. However, quinolones are now emerging as causative agents of pseudotumor cerebri in infants and young children (30).

5 Isolation and antibiotic susceptibility of Salmonella, Shigella, and Campylobacter in Egypt 37 Table 3. Significant changes in the resistance profiles of some enteric pathogens isolated from Egyptian diarrhoea patients during an interval of 4 years before and after 1990 Species 1 Antibiotic a No. of resistant isolates % of total No. of resistant isolates % of total P value Salmonella typhi Am E-07 C E-07 Te E-07 Cb E-05 Salmonella Group B C Group C2 C Cr E-03 S. dysenteriae Te E-04 S. flexneri SxT E-07 S. boydii Te E-07 a Key to antibiotic names: Am=ampicillin, Cr=cephalothin, C=chloramphenicol, Cb=carbenicillin, Te=tetracycline, and SxT=trimethoprim-sulphamethoxazole. P values for differences in susceptibility of other species or serogroups were high at 95% confidence level. We have observed significant differences (p<0.05) in the resistance profiles of some serogroups of Salmonella between late 1980s and early 1990s (Table 3). A similar observation was seen in the Indian subcontinent, Asia, and Africa since 1989 (20,31). Strains of S. typhi, resistant to chloramphenicol, ampicillin, and trimethoprim, have been responsible for numerous outbreaks in these areas (20,22,31). It appears that ciprofloxacin may be the defacto antibiotic of choice for enteric infections. Unfortunately, chromosomallyencoded resistance to ciprofloxacin has now been observed in a small number of strains from the Indian subcontinent, making it of paramount importance to limit its unnecessary use (20,25). This study reports the rates, seasonality, antimicrobial susceptibility, virulence, and regional importance of the pathogens associated with acute enteric infections in Egypt. Data indicate that there is a little difference between our findings and those of the neighbouring countries. Because of the health threat posed by these pathogens, their antibiotic resistance should be continuously monitored (32). Control strategies, such as improving public hygiene, preventing judicious use of existing enteric vaccines and antibiotics, or the development of newer, more broadly protective vaccines, may be more effective in curbing the morbidity and mortality associated with these agents. ACKNOWLEDGEMENTS The authors acknowledge the clinical support of Dr. Nabil Iskander and Dr. Samir Bassily of NAMRU-3. Thanks are also due to Mr. Abdel Hakam Abdel Fattah for expert processing, cultivation, and identification of samples. REFERENCES 1. Jousilahti P, Madkour SM, Lambrechts T, Sherwin E. Diarrhoeal disease morbidity and home treatment practices in Egypt. Public Health 1997;111: Adeleye IA, Adetosoye AI. Antimicrobial resistance patterns and plasmid survey of Salmonella and Shigella isolated in Ibadan, Nigeria. East African Med J 1993;70: Liesenfeld O, Weine T, Hahn H. Three-year prevalence of enteropathogenic bacteria in an urban patient population in Germany. Infection 1993;21: Taylor DN, Bodhidatta L, Echeverria P. Epidemiologic aspects of shigellosis and other causes of dysentery in Thailand. Rev Infect Dis 1991;13:S Blaser MJ, Taylor DN, Feldman RA. Epidemiology of Campylobacter jejuni infections. Epidemiol Rev 1983;5: Moyer NP, Holcomb LA. Campylobacteriosis. In: Balows A, Hausler WJ, Lennette EL, editors. Laboratory diagnosis of infectious diseases: principles and practice. New York: Springer-Verlag, 1988: Butzler JP, Dekeyser P, Detrain M, Dehaen F. Related vibrio in stools. J Pediatr 1973; 82: Zaman R. Campylobacter enteritis in Saudi Arabia. Epidemiol Infect 1992;108: El-Saifi A, Kamel M, Mohi El-Din A, Zagloul I, Podgore JK, Mansour NS et al. Parasitic, bacterial and viral etiology of acute diarrhea in Egyptian children. Med J Cairo Univ 1985;53: Miller P, Hirschhorn, N. The effect of a national control of diarrheal diseases program on mortality: the case of Egypt. Soc Sci Med 1995;40:S1-30.

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