Toledo, Maria Regina Fernandes de

Trabulsi, Luiz R.

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Rev. Inst. Med. trop. São Paulo 11(5):358-362, setembro-outubro, 1969 una 616.98 616. 34-002 ESCHERICHIA GOL! SEROGROUP 0115 ISOLATED FROM PATIENTS WITH ENTERITIS. BIOCHEMICAL CHARACTERISTICS ANO EXPERIMENTAL PATHOGENICITY Luiz R. TRABULSI ( 1 ) and Maria Regina Fernandes de TOLEDO ( 2) SUMMARY Eight strains of E. coli serogroup 0115 isolated from patients with entent1s were studiéd. All strains were capable of causing experimental keratoconjunctivitis in the guinea-pig. Ingestion of one strain by an adult volunteer was followed by a typical dysenteric syndrome, definite signs of an intense inflammatory reaction in the intestinal mucosa, profuse and increasing growth of the ingested strain and a rising titer of serum agglutinins against the O antigen of the strain. ln regard to the biochemical characteristics they were similar to those given by the Escherichia group, but lactose was late fermented or not fermented, lysine was not descarboxilated and the eight strains were non-mobile. One strain did not produce gas from glucose and other sugars. INTRODUCTION The use of the experimental model des-cribed by SERÉNY 7 has permited the disco-very of severa! E. coli serotypes which share with Shigella strains the capacity of causing experimental keratoconjunctivitis in the gui-nea-pig. Most of these serotypes have been found in association with cases of enteritis, both in children and adults 10 • The purpose of this paper is to present the result of studies on E. coli serogroup 0115 strains, which are capable of causing experimental keratoconjunctivitis in the gui-nea-pig and intestinal infection in man. The first strain of the series was isolaLed in 1964 from an adult patient with severe diarrhea and was designated culture 185T-649. As explained further below, culture 185T-64 was first thought to be a new E. coli serogroup 1-0 but later, · upon checking with Dr. F. Orskov, International Escheri-chia Center, Copenhagen, Denmark, all strains of culture 185T-64 were found to belong to Escherichia coli serogroup 0115. MATERIAL AND METHODS Strains - Eight slrains were studied. They were isolated from children and adult pati-ents with acute diarrhea (Table I). ln all cases no recognizable pathogens as Salmo-nella, · Shigella or enteropathogenic E. coli were isolated. Some strains were isolated as nearly pure culture from the stool specimes. The strains were kept in tubes of nutrient agar sealed with paraffined corks. Biochemical tests - These were made as described by CARPENTER et ai. 2, the sodium acetate test being performed as described by TRABULSI & EWING 8 • Pathogenicity to the guinea-pig eye -The test was carried out as described pre-Work of the "Departamento de Microbiologia e Imunologia da Faculdade de Medicina da Univer-sidade de São Paulo (Director: Prof. Dr. Carlos da Silva Lacaz) (1) Professor of Bacteriology. Caixa Postal, 2921 - São Paulo, Brasil (2) Fellowship the "Fundação de Amparo à Pesquisa do Estado de São Paulo". Instituto de Biologia da Universidade de Campinas, São Paulo, Brasil 358 TRABULSI, L. R . .& TOLEDO, M. R. F. de - Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicity. Rev. Inst. Med. trop. São Paulo 11:358-362, 1969. TABLE I Source of strains of E. coli serogroup 0115 and reported manifestations of patients Strains 185T-64 185Tl-64 185T2-65 185T3-65 185T4-65 i85T5-65 185T6-66 Age (years old) 54 27 1 5 2 2 viously 10• ln all cases the infeclion was confirmed hy. bacteriological examinations. F eeding experiment on an adult volunteer - To perform the experiment strain 185T7-66 was selected out of the 8 studied. It had heen isolated a few months ago, from a 3 y. o. child suffering from acute diarrhea. It was sensitive to Neomycin and other drugs. Its virulence to the guinea-pig eye was test-ed hefore the feeding-experiment. The ino-culum was prepared from the growth of the strain on nutrient agar slant incuhated for 18 ,hours at 37ºC. Organisms were harvested in nutrient hroth and 2 mi inoculated into , 60 ml of chilled pasteurized milk. The ino-culum was fed to the volunteer and the -o~mher of viahle cells present in it was de-terminaled by plate counting. The inoculum contained 3,0 X 10 8 viahle cells. The test subject was an adult male, 48 y. o., without symptoms or signs of illness as judged by complete clinical examination. Previous to the experiment, a rectosigmoi-doscopic examination was performed, feces were collected for culture and a hlood sam-ple taken for serum agglutinin determination. Following intake of the inoculum, tempera-ture was measured at 4 hours intervals and symptoms and signs of illness were record-ed. The numher of stools passed was record-ed and fecal samples were taken for mi-Patients Manifesta tions Acute diarrhea for about 24 hours: 15 eva-cuations, abdominal pains, nauseous sensa-tion, illbeing and fever Acute diarrhea: 4 evacuations a day Acute diarrhea: 4 evacuations Acute diarrhea for 2 days; abdominal pains, vomiting Acute diarrhea Acute diarrhea; fever (38,5°) Acute diarrhea; fever croscopical and bacteriological examination. A second rectosigmoidoscopic examination was performed 24 hours after the inoculation and hlood sample for serum agglutinin de-termination taken 15 days !ater. After treatment dayly fecal samples were collected for bacteriological examination during 3 days. The feces cultures were made hy seeding the specimes in McConkey and SS agar, and the suspected colonies identified hy bio-chemical and serological tests. RESULTS Biochemical characteristics - The results of biochemical tests are shown in Table II. The reactions of the 8 strains were homoge-nous in most of the tests. However there was some strain variation in the fermentat-ion of lactose, salicin, maltose, rhamnose and in ONPG tests. It should be noted that strain 185T-3 did not produce gas in any of the fermented carhohydrates and strain 185T-5 neither fermented lactose nor gave a positive ONPG reaction. Pathogenicity to the guinea-pig eye -The 8 strains caused keratoconjunctivitis m the guinea-pig, in all aspects identical to that caused hy virulent Shigella strains. ln 359 TRABULSI, L. R. & TOLEDO, M. R. F. de - Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicit:y. Rev. Inst, Med. trop. São Paulo 11:358-362, 1969. TABLE II Biochemical characteristics of the 8 strains of E. coli serogroup 0115 Substract 1185T - 185Tl - 185T2 185~-4 - 185T6 - 185T7 185T3 185T5 Indol VM VP Citrate (Simmons) H 2S Urease Phenylalanine deaminose KCN Gelatin Malonate Lysine decarbox:ylase Arginine dib:Ydrolase Ornithine decarboxilase Motility Christensen's citrate Sodium acetate ONPG Glucose + + + + + AG + + + + + + + + + A AG Lactose AG/(AG)/(Ag) (A) Sucrose Mannitol AG A AG Dulcitol AG A AG Salicin A/Ag/ag (A) (Ag) Adonitol Inositol Sorbitol AG A AG Arabinose AG A AG Raffinose Rhamnose A/(A)/(a) (a) (a) Maltose AG/(AG)/(ag) A AG X:ylose Trehalose Cellobiose Dextrin Starch Nitrate Oxidase A a := Strong acid Weak acid AG A AG AG A AG + + + G g ( := := ) Gas 1/4 or more than the volume of Durhan tube Gas less than 1/4 the volume of Durhan tube Acid and gas after 48 hours all of the cases the inoculated strain was demonstrated in the ocular secretion in great amounts during the first days of the disease. Feeding experiment on an adult volunteer - The pre-feeding examination gave the following results: feces culture showed no pathogens, agglutinins against a boiled sus-pension of strain 185T-7 was not found at the 1/4 dilution of the serum and the recto-360 sigmoidoscopical examination showed a nor-mal mucosa. Six hours after the inoculation the volun-teer became ill and the illness was allowed to take its course for 18 hours. The volunteer was then trated with Neomycin and antis-pasmodics. Following the treatment, he felt better and only one further liquid evacuation was passed. ln the 3 feces culture perform-TRABULSI, L. R. & TOLEDO, M. R. F. de - Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicity. Rev. Jnst. Med. trop. São Pa1.Zo 11: 358-362, 1869. ed in the subsequent 3 days, strain 185T-7 was not found. The predominant symptoms were diarrhea, abdominal cramps, anorexia and weakness. The last two manifestations were more in-tense in the second day following inoculation. No rise in temperature was recorded. A total of 9 evacuations were passed in the 18 hours period. With exception of the first evacuation, feces were liquid, mucous, bloody and contained a great number of leukocytes. Bacteriological examinations of stool spe-cimes collected from the lst., 2nd., 3rd., 4th., 7th., 8th., and 9th. evacuations, showed a profuse and increasing growth of strain 185T-7 in both media used. ln SS medi um, the strain grew as a nearly pure culture from the second evacuation on. The second rectosigmoidoscopic examinat-ion showed that the mucosa was congested a:nd edematiated, bleeding easily. The rec-tosigmoidoscopy did not progress beyond 14 cm. The serum agglutinin titer against a heat-ed suspension of strain 185T-7 raised to 1 :84 at the second determination. DISCUSSION As .above mentioned, culture 185T-64 was fírst thought to be a new E. coli serogroup 10 • W e carne upon this conclusion beca use the str11-in was not significantly agglutinated by any of the 01 to 0146 E. coli sera and Dr. W. H. Ewing (Enteric Unit, CDC, Atlanta, Ga.) had identified is as "E. coli O group undeterminated". The different results ob-tained in the two first laboratories and in the lnternational Escherichia Center are well explained in a recent paper by GLANTZ et al. 4 who observed' the sarne kind of dis-érepancy. The controversial results were due to a mixup in the lnternational Salmonella in the distribution of the standard E. coli serogroup 0115, in 1950. As a consequence, many laboratories, instead of having the standard strain and antiserum for E. coli serogroup 0115, have actually another strain only serologically related to the standard E. coli 0115. E. coli serogroup 0115 was originally iso-lated by WRAMBY, in Sweden from calves with septicemia. Since then, it has been rather common in different animais diseases, specially in septicemias in calves in Euro-pe 5 • GLANTZ et al. 4 also reported infection in cattle by these organisms in the U. S. A. , and confirmed experimentally its pathogeni-city for colostrum-deprived calves. The present studies add to our knowledge on the pathogenicity of E. coli serogroup 0115. It has been shown that all the strains isolated from man did cause experimental keratoconjunctivitis in the guinea-pig, and their capacity to cause intestinal infection in man seems to have been proved beyond doubt. Following the ingestion of the ino-culum the volunteer develop a typical dysen-teric syndrome, with a profuse and increas-ing growth of the ingested strain, a rising titer of serum agglutinins against the O an-tigens of the strain and definite signs of an intense inflammatory reaction in the intestinal mucosa, as shown by the recto-sigmoidoscopic examination and the presen-ce of blood and pus in feces. Furthermore, all strains were isolated from patients with acute enteritis and in studies which are being carried out we have not found these organisms in the feces of more than 200 normal persons so far tested. However it should be stressed that the present findings cannot be extend to E. coli serogroup 0115 as a whole. A comparative study between the strains isolated in our laboratory and the strains isolated from cal-ves has been not carried out. The latter are typical E. coli strains 6 and is not im-possible that both gr0ups of strains have non-identical O or K antigens as well as different pathogenic properties. lt is worth to mention that the manifes-tations presented by the volunteer and the capacity of the strains to cause intestinal in-fection in adults and experimental kerato-conjunctivitis in the guinea-pig indicate that their pathogenic properties are different from those of the classic enteropathogenic E. coli serotypes responsible for infantile enteritis. lt is more probably that they, as well as others E. coli strains virulent to the guinea-pig eye, are very dose or identical to Shigella in regard to pathogenicity. ln re-361 TRABULSI, L. R. & TOLEDO, M. R. F. de - Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicity. Rev. Jnst. Med. trop. Sã.o Paulo 11:358-362, 1969. gard to this, it is significant that Dr. P. CARPENTER ( Disentery Reference Laboratory - Colindale, London) found out by cross absorption tests that strains 185 are strong related if not identical to the provisory Shigella type 3341-556 1 • Most of the others E. coli serotypes capa-ble of causing experimental keratoconjuncti-vitis in the guinea-pig also have O antigens identical to those of certain Shigella sero-types. This is the case with E. coli serotypes 032, 0124, 0143 aríd so on 3 • ln regard to the biochemical characteris-tics, it should be pointed out that different biotypes were found. lt is also noteworthy that lactose was late fermented or not fer-mented and that ali the strains were non-motile and did not descarboxylate lysine. The latter seems to be a common characte-ristics to all Escherichia coli serotypes ca-pable of causing experimental keratoconjunc-tivitis in the guinea-pig 10 • RESUMO Escherichia coli do grupo sorológico 0115 isolado de pacientes com enterite. Caracte-rísticas bioquímicas e patogenicidade expe-rimental Oito amostras do colibacilo 0115, isoladas de pacientes com enterite, foram estudadas. Tôdas causaram ceratoconjuntivite experi-mental no cobaio. A ingestão de uma amos-tra por um voluntário adulto foi seguida de síndrome disentérica típica, sinais de rea-ção inflamatória intensa na mucosa intes-tinal, proliferação ascendente da amostra e aumento do título de anticorpos séricos con-tra o antígeno O da amostra ingerida. Bio-quimicamente, as oito amostras se compor-taram como Escherichia, mas a lactose foi iermentada tàrdiamente por 7 amostras, e não fermentada por uma amostra. Nenhu-ma amostra descarboxilou a lisina e as oito foram iméveis. Uma amostra não produziu gás dos carboidratos fermentados. ACKNOWLEDGMENTS W e wish to thank Dr. K. P. Carpenter and F. Orskov for the studies performed 362 and for reading this paper and Dr. W. H. Ewing for the serological tests made with the first strain. REFERENCES 1. CARPENTER, K. P. - Personal communi-ca tion, 1968. 2. CARPENTER, K. P.; LAPAGE, S. P. & STEEL, K. J. - Biochemical identification of Enterobacteriaceae. ln GIBBS, B. M. & SKINNER, F. A. - Jdentificati.on Me-thods for Microbiologists. London, Academic Press, 1966, pp. 21-33. 3. EDWARDS, P. R. & EWING, W. H. Jdent(fi.cation of Ente,·obacteriaceae. 2nd. Edition. Atlanta, Burgess, 1962. 4. GLANTZ, P. J.; KRADEL, D. C. & HO-KANSON, J. F. - Escherichia coli sero-group 115 isolated from animals: experi-mental infections of calves. Amer. J. Vet. Res. 27 .1205-1209, 1966. 5. ORSKOV, F. Antigenic rela tionship between O groups 1-112 of E. coli and Wramby's O groups 26w-43w. 10 new O groups: 114-123. Acta Pa.th. Microbial. Scand. 31: 51-56, 1952. 6. ORSKOV, F. - Personal communication, 1969. 7. SERÉNY, B. - Experimental keratocon-junctivitis shigellosa. Acta Microbial. Himg. 4:367-376, 1957. 8. TRABULSI, L. R. & EWING, W. H. -Sodium acetate medium for the differentia-tion of Shigella and Escherichia coli cultu-res. Pub. Health La/1. 20:137-140, 1962. 8. TRABULSI, L. R.; ZULIANI, M. E. & SERRANO, J. A. - On two new en tero-bacteria pathogenic to the guinea-pig eye (Cultures 185T-64 and 193T-64). Rev. Inst. Med. trop. São Paulo 7:241-246, 1965. 10. TRABULSl, L. R.; FERNANDES, M. R. F. & ZULIANI, M. E. - Novas bactérias pa-togênicas para o intestino do homem. Rev. Jnst. Med. trop. São Paulo 9:31-39, 1967. Recebido para publicação em 29/4/1969. 

Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicity

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Escherichia coli serogroup 0115 isolated from patients with enteritis. Biochemical characteristics and experimental pathogenicity

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