The Prevalence of Enteropathogenic Escherichia coli in Infantile Diarrhea and their Antibiotic Susceptibility Pattern

Illuminado P. Livelo*
(*Department of Microbiology, Research Institute for Tropical Medicine, Department of Health, Alabang, Metro Manila)

ABSTRACT
Diarrheal disease is a major cause of illness and death among infants, and young children worldwide. In the study of diarrhea in man, especially in young infants, the principal microorganisms implicated are salmonella, shigella and enteropathogenic strains of Escherichia coli (EEC). The objective of this study was to determine the prevalence of enteropathogenic E. coli in infantile diarrhea using as subjects children with ages ranging from birth to two years. All were suffering from diarrhea when admitted at the Philippine General Hospital. The most prevalent serological type of enteropathogenic E. coli isolated from the stools of diarrheic patients and the antibiotic susceptibility pattern were also determined. The study yielded the following results: During the 13 month period (Feb. 1, 1976 - Feb. 28, 1977), 1,607 infants suffering from diarrhea were found to be due to specific pathogenic organisms such as EEC, salmonella and shigella. Eighty three or (5.2%) were found to harbor EEC. The remaining 1,252 infants were found to harbor "normal flora" of the gastrointestinal tract. No viral studies were done. Among the E. coli strains isolated, serotype 0119: K 69 (B 14) was most frequently seen (54.2%), followed by 0111: K 58 (B 4) (27.7%), 055: K 59 (B 5) (7.2%), 026: K 60 (B 6) and 0128; K 67 (B 12) (3.6%), 086a: K 61 (B 7) (2.4%) and lastly 0126: K 71 (B 16) (1.2%). E. coli serotypes 0119: K 69 (B 14) and 0111: K 58 (B 14) were both sensitive to gentamicin, cephalothin, trimethoprim sulfonamide and furadantin and resistant to streptomycin, tetracycline and ampicillin. The other E. coli serotypes showed multiple resistance to antibiotics. Since the data from this study were collected in 1977-78 comparison with current and future surveillance studies on enteropathogenic E. coli and its antibiotic susceptibility patterns will provide information as to what significant changes are occurring in this particular bacterial species. [Phil J Microbiol Infect Dis 1987; 16(2): 54-58] Key Words: E. coli, enteropathogenic, diarrhea, salmonella, shigella

INTRODUCTION In the study of diarrhea in man, especially in young infants, the principal microorganisms implicated are salmonella, shigella and enteropathogenic strains of Escherichia coli. The association of certain strains of enteropathogenic Escherichia coli with infantile diarrhea was suspected as early as 1923. 8 In 1945, Bray was able to associate a specific antigenic type of Bacterium coli with infantile diarrhea. B. coli was the earlier term for enteropathogenic Escherichia coli, The following serotypes of E. coli are now generally accepted as etiological agents of diarrheal disease in infants and children (0 to 2 or 2.5 years of age): 026: K 60 (B6), 055: K 59 (B5), 086a: K 61 (B7), 0111: K 58 (B4), 0119: K 69 (B14), 0125: K 70 (B15), 0126: K 71 (B16), 0127: K 63 (B8), 0128:K 67 (B12), 0112: K 68 (BI3), 0124: K 72 (B17). The enteropathogenic strains of E. coli are characterized by 2 factors: the ability to colonize the jejunum and the ability to cause movement of water and electrolytes across the intact intestinal epithelium into the bowel lumen, without either local or systemic invasion of the organism. 8 Such movement is called enterosorption, which results from the production of an exotoxin known as enterotoxin. In turn, the capacity to produce enterotoxin is determined by a plasmid (or episome), designated as the "resistance transfer factor" or RTF, which can be transmitted from one strain of E. coli to another and to other species of bacteria by sexual conjugation. 3,8 Hence, it has been suggested that the reason why certain serotypes of E. coli are enteropathogenic with fair regularity is that they are good plasmid receptors. Attached to the RTF are genes for resistance to different drugs. This would explain the occurrence of multiple

resistance among enteropathogenic E. coli. Aragon et al1 made a study of E. coli serotypes associated with diarrheal conditions in Manila and their survey yielded the following results: 1) 63 of 502 patients or 12.5% were found to harbor enteropathogenic E. coli; 2) the four most common EEC serotypes were 026: K 60 (B6), 055: K 59 (B5), 0111: K 58 (B4) and 0128: K (BI2). 0111: K 58 (B4) together with 0127: K 63 (BS) were also found in the study by Solomon et al7 to be the most frequently isolated strains of EEC. In the study conducted by Kaslow et al,4 in Alabama, 14 of 92 infants suffered from mild diarrhea and 12 of these had specimens positive for E. coli 0128: K 67 (B12). All isolates were found susceptible to neomycin, colistin sulfate, kanamycin, tetracycline hydrochloride, sulfathiazole, streptomycin sulfate, chloramphenicol, nitrofurantoin, nalidixic acid and gentamicin sulfate. All but one were susceptible to cephalothin sodium and ampicillin sodium. In 1960-1961, a study of the etiology in infantile diarrhea was conducted in Houston8 and the most commonly defined pathogen during this period was E. coli 0111: K 58 (B4), with a remarkable pattern of multiple antibiotic resistance. It was recognized that this E. coli strain was resistant to chloramphenicol and tetracycline as well as to neomycin and kanamycin. Colistin sulfate was the drug of choice during this period. The purpose of the current study was to survey the prevalence of enteropathogenic Escherichia coli in infantile diarrhea locally. It aimed to: 1) determine and identify the different serologic types of enteropathogenic Escherichia coli isolated from the diarrheic infants; 2) determine the most prevalent enteropathogenic Escherichia coli serotype; and 3) determine the antibiotic susceptibility pattern of the isolates. MATERIALS AND METHODS The Study group consisted of infants with diarrhea admitted at the Philippine General Hospital, with ages ranging from birth to two years. Specimens were obtained from all patients during a 13-month period (Feb. 1, 1976 to Feb. 28, 1977). This was carried out as part of the routine ad mission procedure. Rectal swabs or stool cultures were obtained from all infant patients. The specimens were placed in sterile tubes or bottle s and inoculated within 30 minutes onto the surface of MacConkey agar, Salmonella -Shigella agar, Selenite F broth, alkaline Peptone, and phenylethanol agar and then incubated at 37C for 18 to 24 hours. The primary plating media were examined for colonies of E. coli. The organisms were then inoculated on Triple sugar iron agar, Lysine iron agar, Simmon Citrate agar, SIM medium, Christensen agar, and Phenylalanine deaminase to further substantiate the verification of E. coli isolates. After identification, the E. coli isolates were suspended in saline and mixed on a" slide with commercial E. coli typing serum. Enteropathogenic E. coli gave a 4+ agglutination immediately in type-specific serum. The specific antisera employed were: 0111:K 58 (B4), 055: K 59 (B5), 026:K 60 (B6), 0127a:K 63 (BS), 086a:K 61 (B7), 0128:K 67 (B12), 0119:K69 (B14), 0125:K 70 (B15), 0126:K 71 (BI6) and 0124:K 72 (BI7). E. coli serology was based on the presence or absence of three kinds of antigens: A, L and B, all of which mask the reaction and must be destroyed by heat before agglutination in 0 antiserum takes place. The standardized antibiotic sensitivity test used in this study was the disc diffusion test,2 which was carried out according to the technique of Kirby-Bauer using a "single high potency" disc. The Kirby-Bauer technique included the use of a inoculum comparable to that of a turbidity standard prepared by adding 0.5 ml of 0.048 M BAC 12 to 99.5 ml of 0.36 N H2 SO4 which is approximately equal to the turbidity due to 108 Enterobacteriaceae/ml; the use of Mueller Hinton medium with pH 7.2 to 7.4 which supports the growth of most organisms; and the use of one antibiotic disc of high potency to represent an antibiotic group. 6 No viral studies were performed.

The following schema outlines the cultivation, biochemical and serological methods employed in the isolation and identification of E. coli.
Specimen

Primary Plating Salmonella-Shigella Agar Selenite F broth Alkaline Peptone TCBS Phenylethanol Agar Serology E. coli OK Antisera slide agglutination with heated and unheated culture Serotyping with the following antisera 0111 : K58 (B4) 055 :K59 (BS) 026 : K60 036) 086 :K61, (B7) 0127a :K63 (B8) 0124 : K72 (BI7) 0125 : K70 (B15) 9126 :K71 (BI6)

MacConkey Agar

Biochemical Tests Lysine decarboxylase Triple sugar iron Simmon citrate Motility Urease Indole H2S Phenylalanine deaminase Interpretation: d - variable A - acid G - Gas

d A/AG + + -

RESULTS AND COMMENTS: The stool examinations of a total of 1,607 diarrheic infants admitted at the Philippine General Hospital, with ages ranging from birth to two years, were evaluated. Table 1 shows the number of infants suffering from diarrhea who were harboring normal bacterial flora of the gastrointestinal tract, those suffering from diarrhea due to pathogenic organisms other than enteropathogenic E. coli and those infants suffering from diarrhea due to enteropathogenic E. coli. The different strains found in patients suffering from diarrhea due to organisms other than enteropathogenic E. coli are shown in Table 2. Identification of the serologic types of enteropathogenic E. coli isolated from the stool of diarrheic patients is shown in Table 3.
Table 1. Distribution of Diarrheic Infants by Types of Organisms Isolated Organisms Normal Flora* Pathogenic organism other than E. coli Enteropathogenic E. coli Total *No enteropathogenic organism isolated No. of Infants 1,252 272 84 1,607 Percentage 77.9 16.9 5.2 100.0

Of the 45 infants harboring 0119:K69 (B14) strain, 4 were also harboring salmonellae at the same time. One harbored Citrobacter freundii together with 0111:K58 (B4) strain, 2 had 011: K58 (B4) together with salmonellae and 1 had 055:K59 (B5) together with salmonellae. Apparently, the most prevalent enteropathogenic E. coli strain found in this study was 0119:K69 (B14) (54.2%) followed by 0111:K 58 (B4) (27.7%). In the study conducted by South8 in Chicago and in Houston, the most commonly defined pathogen was E. coli 0111:K 58 (B4), 7 while in the study done by Solomon et al in Boston, the most frequently isolated strains of EEC

were 0127:K 63 (B8) (36%) and 0111:K 58 (B4)(25%). In the study conducted in Manila by Aragon et al, the four most common EEC serotypes were 026:K 60 (B6), 055:K 59 (B5), 0111:K 58 (B4) and 0128:K 67 (312).
Table 2. Frequency of Occurrence of other Pathogenic Organisms Strain Salmonella Klebsiella pneumoniae Citrobacter freundii Arizona Shigella flexneri Yersinia enterocolitica Vibrio cholera Staphylococcus aureus Total No. of Infants 204 23 21 16 3 1 1 3 272 Percentage 75.0 8.4 7.7 5.9 1.1 0.4 0.4 1.1 100.0

Table 3. Frequency Distribution of Enteropathogenic E. coli Serotype 0119:K69(B14) 0111:K58(B4) 055:K59(B5) 026:K60(B6) 0128:K67(BI2) 086a:K61(B7) 0I26:K71(BI6) Total No. of Infants 45 23 6 3 3 2 1 83 Percentage 54.2 27.7 7.2 3.6 3.6 2.4 1.2 100.0

Enteropathogenic E. coli isolated from the stool specimens were subjected to different antibiotic susceptibility testing. Among the antibiotics used were cephalothin (CR), ampicillin (AM), kanamycin (K), gentamicin (GN), chloramphenicol (C), tetracycline (TE), trimethoprim/sulfonamide (SXT), furadantin (FM), streptomycin (STREP), neomycin (NEe) and colistin sulfate (CL). During the 13-month period, not all the antibiotics were available all the time. This accounts for a number of enteropathogenic E. coli isolates which were not subjected to some of these antibiotics. The antibiotic susceptibility of the different enteropathogenic E. coli strains is shown in Table 4. Because the predominant serotype of E. coli isolated were 0119: K 69 (B14) and 0111: K 58 (B4), the percentage number of these strains showing multiple resistance was determined. Tables 5 and 6 show the response of E. coli 0119: K 69 (BI4) and E. coil 0111: K 58 ([14) to the 11 antibiotics used in the study. Of the 45 infants harboring E. coil 0119: K 69 (B14), 34 were tested for streptomycin effect and were all found to be resistant to it. 11 were resistant to streptomycin, tetracycline and ampicillin, 9 were resistant to streptomycin, neomycin and ampicillin, 9 were resistant to streptomycin, kanamycin and ampicillin and 4 were resistant to streptomycin, neomycin as well as to ampicillin and kanamycin. Twenty four infants were found resistant to ampicillin and streptomycin. There were 23 infants harboring E. coil 0111: K 58-(B4). Eleven of them were found resistant to ampicillin and streptomycin, 10 were resistant to ampicillin and neomycin, 6 were resistant to neomycin, streptomycin and tetracycline, 5 were resistant to ampicillin, tetracycline and streptomycin and 4 were resistant to ampicillin, tetracycline, streptomycin and neomycin. Tables 5 and 6 seem to agree with the finding that several serotypes of E. coli had multiple antibiotic resistance.

Table 4. Antibiotic Susceptibility of Enteropathogenic E. coli Isolates

0119: (B14) S R 19 90.5 5 13.5 2 16.7 20 96.6 2 10.0 1 5.3 31 89.7 13 100. 0 34 100. 0 14 58.3 2 9.5 32 86.5 10 83.3 1 3.4 18 90.0 18 94.7 4 10.3 14 87.5 4 100. 0 1 5.9 1 6.2 3 60.0 0111: (B4) S R 7 87.5 3 16.7 1 50.0 11 100. 0 4 28.6 1 12.5 15 83.5 1 50.0 055: (B5) S R 4 75.0 4 66.7 1 100. 0 5 100. 0 3 100. 0 2 50.0 4 100. 0 3 100. 0 2 100. 0 2 100. 0 1 100. 0 1 12. 5 2 33. 3 026: (B6) S R 1 100. 0 1 100. 0 0118: (B12) S R 1 33.3 2 66.7 3 100. 0 1 100. 0 2 100. 0 2 100. 0 2 100. 0 1 33.3 2 66.7 1 100. 0 1 100. 0 086: (B7) S R 1 100. 0 1 100. 0 1 100. 0 1 100. 0 1 100. 0 1 100. 0 0216: (B16) S R 1 100. 0 1 100. 0

Antibiotic s CR AM K GN C TE SXT FM STREP NEO CL

2 66.7

1 33.3

10 71.4 10 100. 0 2 12.5

2 50. 0 3 100. 0 1 100. 0 1 33.3 1 33.3

1 100. 0

1 100. 0

2 66.7 2 66.7 2 66.6 1 33.3 1 100. 0

16 94.1 15 93.8 2 40.0

10 41.7 6 100. 0

S = Sensitive R = Resistant

It appears from Table 6 that the predominant E. coli strains O119:K 69 (B14) and 0111: K 58 (B4) were both sensitive to gentamicin, cephalothin, trimethoprim/sulfonamide and furadantin, and both were resistant to streptomycin, tetracycline and ampicillin. In addition, 0119:K 69 (B14) alone seemed to be resistant to chloramphenicol and kanamycin and sensitive to colistin sulfate, while 0111:K 58 (B4) alone was apparently resistant to neomycin. Gentamicin has been used successfully in the treatment of diarrhea due to enteropathogenic E. coli. This study showed that both 0119:K 69 (B14) and 0111: K 58 (B4) were also sensitive to gentamicin.
Table 5. Percentage Number of Escherichia coli 0119:K69(B14) showing Multiple Resistance Resistance to >7 6 5 4 3 <2 Number 0 2 2 13 11 10 Percentage 0 4.4 4.4 28.9 24.4 22.2

Table 6. Percentage Number of Escherichia coli 0111:K58 (B4) Showing Multiple Resistance Resistant to 6 Number 0 Percentage 0

5 4 3 2 1

2 8 6 5 2

8.7 34.8 26.1 21.7 8.7

The epidemiological studies conducted by South8 and Kaslow et al4 as well as the results of this study, have shown that there is no dependable antibiotic sensitivity pattern as far as the enteropathogenic E. coli are concerned. Necessity for constant surveillance of antibiotic susceptibility of enteropathogenic E. coli is suggested. CONCLUSION A study of 1,607 patients (newborn to two years of age) who were suffering from diarrhea admitted at Philippine General Hospital showed that 355 infants had diarrhea due to pathogenic bacteria, and the remaining 1,252 had normal flora of the gastrointestinal tract. Of the 355 patients suffering from diarrhea due to some pathogenic organisms, 83 or 23.4% were harboring enteropathogenic E. coli. The most prevalent serotype of E. coli was 0119:K 69 (B14) followed by 0111:K 58 (B4). As to the antibiotic susceptibility of the isolates, 0119:K 69 (B14) and 0111:K 58 (B4) were both sensitive to gentamicin, cephalothin, furadantin and trimethoprim/sulfonamide. Both were resistant to streptomycin, tetracycline and ampicillin. Multiple antibiotic resistance was seen in both 0119:K 69 (BI4) and 0111:K 58 (B4) serotypes. It is recommended that the results obtained in this study be tested further. First, there was no control done on the subjects studied; secondly, no follow-up was done; and thirdly, the enteropathogenic E. coli isolates were not all tested to the different antibiotics. However, the data presented provides information for subsequent seroprevalence and antibacterial susceptibility pattern comparisons. Acknowledge ment
This study was done at the Institute of Public Health, University of the Philippines.

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. Aragon PR et al. E. coli serotypes associated with diarrheal conditions in Manila. JPMA 1959; 35:20-24. Bauer AW, Kirby WMM, Sherries JC, Turck M. Antibiotic susceptibility testing by standardized single disc method. Am J Clin Pathol 1986; 45:493. Davis BD, Dulbecco R. Eisen RN, Ginsberg HS, Wood WB Jr. Principles of Microbiology and Immunology. New York: Harper and Row, 1974. Kaslow RA, Taylor A Jr. Eweck NS, Bobo RA, Steele LD, Cassady G Jr. Enteropathogenic Escherichia coli infection in a newborn nursery. Am J Dis Child 1974; 128:791-801. Lennette EH, Spaulding EN, Truant JP. Manual of Clinical Microbiology. American Society for Microbiology: Washington, D.C., 1974. NCCLS: Subcommittee in Antimicrobial Susceptibility Testing Performance Standard for Antimicrobial Disc Susceptibility Test as Used in Clinical Laboratories (Revised Tentative Standards, 2525, W/8th St., L.A. Calif., 1973. Solomon P, Weinstein L, Joress SM. Studies of the incidence of Enteropathogenic Escherichia coli in a pediatric population. J Pediatr1961; 58:716-721. South W. Enteropathogenic Escherichia coli disease: New developments and perspectives. J P ediatr 1971; 79:1 -11.