FREQUENCY AND DISTRIBUTION OF DIARRHOEAGENIC ESCHERICHIA COLI STRAINS ISOLATED FROM PEDIATRIC PATIENTS WITH DIARRHOEA IN BOSNIA AND HERZEGOVINA

Diarrhoeal disease is a major cause of illness and death among infants and young children worldwide. Among the Escherichia coli (E. coli) causing intestinal diseases, there are six well-described categories: enteroaggregative E. coli (EAEC), diff usely adherent E. coli (DAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and enterotoxigenic E. coli (ETEC). Th e aim of the present study was to investigate the relative contribution of diff erent groups of diarrhoeagenic E. coli (DEC) in paediatric patients with diarrhoea. Clinical stool specimens from  children with diarrhoea, with ages ranging from birth to <  years, were selected for the study over a period of  months (August  to December ). Th e study showed that / children () had diarrhoea due to diarrhoeagenic E. coli. Th e most prevalent was enteropathogenic E. coli (EPEC) isolated from / paediatric patients (), followed by enterotoxigenic (ETEC) isolated from / (.), enterohaemorrhagic (EHEC) from / (.) and enteroinvasive (EIEC) from / patients (.). Th e most prevalent serotypes of EPEC were O:K and O:K isolated from / (.), O:K from / patients (), followed by O:Kand O:K isolated from / patients (.). Among the ETEC the most prevalent serotypes were O:K isolated from / (.) and O:K from / patients (.), especially during the fi rst twelve months: / patients (.). Th e most prevalent EHEC strain found in this study was O:Kand O:K-: / patients (.). Two isolated strains of EIEC belong to serotype O:K-. Th e average age of the patients was  years. Two patients with bloody diarrhoea had EHEC serotype O:H which progressed to haemolytic-uremic syndrome (HUS). FREQUENCY AND DISTRIBUTION OF DIARRHOEAGENIC ESCHERICHIA COLI STRAINS ISOLATED FROM PEDIATRIC PATIENTS WITH DIARRHOEA IN BOSNIA AND HERZEGOVINA Amela Dedeić-Ljubović*, Mirsada Hukić, Daria Bekić, Amra Zvizdić Institute of Clinical Microbiology, Clinical Centre of the University of Sarajevo, Bolnička ,  Sarajevo, Bosnia and Herzegovina * Corresponding author


Introduction
Diarrhoeal diseases constitute a major public health problem, particularly in the developing world where the rate of mortality and morbidity is very high.E. coli is the type species of the genus Escherichia that contains mostly motile Gram-negative bacilli that fall within the family Enterobacteriaceae.It is the predominant non-pathogenic facultative fl ora of the human intestine.Th e organism typically colonizes the infant gastrointestinal tract within hours of life, and thereafter E. coli and the host derive mutual benefit ().E. coli usually remains harmlessly confi ned to the intestinal lumen; however, in the debilitated or immunosuppressed host, or when gastrointestinal barriers are violated, even normal "non-pathogenic" strains of E. coli can cause infection ().E. coli that cause diarrhoea are extremely common worldwide.Th e classifi cation of diarrhoeagenic E. coli strains is based on their virulence properties, and comprises six groups: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohaemorragic E. coli (EHEC), enteroaggregative E. coli (EAEC) and diff use adhering/diff usely adherent E. coli (DAEC).Th e associated clinical pictures comprise childhood and traveller's diarrhoea (ETEC), bloody diarrhoea and haemolyticuremic syndrome (EHEC), infantile diarrhoea (EPEC), and bacillary dysentery-like diarrhoea (EIEC) (, ).ETEC are associated with two major clinical syndromes: weanling diarrhoea among children in the developing world, and traveller's diarrhoea.Certain serotypes of ETEC occur worldwide; others have a limited recognized distribution.Epidemiologic investigations have implicated contaminated food and water as the most common vehicles for ETEC infection (, , , and ).ETEC diarrhoea may be mild, brief, and self-limiting or may result in severe purging similar to that seen in V. cholerae infection ().EPEC is an important category of diarrhoeagenic E. coli which has been linked to infant diarrhoea in the developing world.As with other diarrhoeagenic E. coli strains, transmission of EPEC is faecal-oral, with contaminated hands, contaminated foods, or contaminated fomites serving as vehicles.EPEC adhere to the mucosal cells of the small bowel.The result of EPEC infection is watery diarrhoea, which is usually self-limited, but can be chronic ().EHEC can be transmitted by food and water and from person to person.Most cases are caused by ingestion of contaminated foods, particularly foods of bovine origin ().In most patients, the bloody diarrhoea will resolve without apparent sequelae, but in about  of patients younger than  years (and in many elderly patients), the illness will progress to HUS.HUS is defi ned by a trial of haemolytic anaemia, thrombocytopenia, and renal failure; initial clinical manifestations include oligouria or anuria, oedema, pallor, and, sometimes, seizures ().There are some biochemical characteristics of E. coli O:H that have been exploited in the isolation and identifi cation of this serotype.An important characteristic is that O:H strains do not ferment D-sorbitol rapidly, in contrast to about  to  of other E. coli strains ().
Enteroaggregative E. coli (EAEC) is a subgroup of diarrhoeagenic E. coli (DEC) that during the past decade has received increasing attention as a cause of watery diarrhoea, which is often persistent.EAEC have been isolated from children and adults worldwide.As well as sporadic cases, outbreaks of EAEC-caused diarrhoea have been described.Th e defi nition of EAEC is the ability of the micro-organism to adhere to epithelial cells such as HEp- in a very characteristic 'stacked-brick' pattern.Typical illness is characterized by watery, mucoid, secretory diarrhoea with low-grade fever and little to no vomiting.However, up to one third of patients with EAEC diarrhoea had grossly bloody stools (, ).EIEC strains are biochemically, genetically, and pathogenetically related closely to Shigella spp.Both organisms have been shown to invade the colonic epithelium, a phenotype mediated by both plasmid and chromosomal loci.In addition, both EIEC and Shigella spp.elaborate one or more secretory enterotoxins that may play roles in diarrhoeal pathogenesis ().EIEC infection presents most commonly as watery diarrhoea, which can be indistinguishable from the secretory diarrhoea seen with ETEC.Only a minority of patients experience the dysentery syndrome, manifested as blood, mucus, and leukocytes in the stool, tenesmus, and fever.In two documented EIEC outbreaks, gross blood was observed in  and  of persons infected (, ).Asymptomatic infections due to EIEC are probably unusual.
Our study shows that diarrhoeagenic E. coli is a signifi cant causal agent of diarrhoeal diseases in paediatric patients in Bosnia and Herzegovina.Th is study is the fi rst report about the frequency and most common serotypes of DEC in Bosnia and Herzegovina.Additionally, it is the fi rst report of cases with an O:K-infection which progressed to HUS, a serious and potentially fatal illness.KEY WORDS: diarrhoeagenic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, enterohaemorrhagic E. coli, enteroinvasive E. coli, haemolytic uremic syndrome.().
In , Kauffman proposed a scheme for the serologic classifi cation of E. coli which is still used in modifi ed form today.According to the modifi ed Kauff man scheme, E. coli is serotyped on the basis of their O (somatic), H (fl agellar), and K (capsular) surface antigen profiles.A total of  different O antigens, each defi ning a serogroup, are recognized currently.A specifi c combination of O and H antigens defi nes the serotype of an isolate.E. coli of specifi c serogroups can be associated reproducibly with certain clinical syndromes, but it is not in general the serologic antigens themselves that confer virulence.Rather, the serotypes and serogroups serve as readily identifi able chromosomal markers that correlate with specific virulent clones (, ).

Patients and stool samples
 children with acute diarrhoeal diseases, admitted to the Paediatric clinic, Clinical Centre of the University of Sarajevo, were included.The age distribution ranged from birth to <  years with a mean age of  years.A total of  stool samples were examined for the presence of diarrhoeagenic E. coli.Stools were collected in plastic containers.

Stool culture
Stool culture was done at the Institute of Microbiology, Clinical Centre of the University of Sarajevo.E. coli was recovered from clinical stool specimens on MacConkey agar (Becton Dickinson).Th e plates were incubated overnight at °C (±.°C) in bacteriological incubators under aerobic conditions.Th is media is specially designed to distinguish lactose-fermenting (pink to red) from nonlactose-fermenting colonies (colourless or slightly beige).

Biochemical identifi cation
Enterobacteriaceae are usually identifi ed via biochemical reactions.Th ese tests were performed in individual culture tubes, while the indole test, positive in  of E. coli strains, was used for the diff erentiation from other members of the Enterobacteriaceae.Fermentation of D-sorbitol was used for the diff erentiation of EHEC O:K-.

Serotyping
Bacterial growth on a primary-culture plate was agglutinated by slide agglutination with polyspecifi c O-antisera, Anti coli I (O, O, O, O, O, and O), Anti coli II (O, O, O, O, O, O, and O) and Anti coli III (O, O, O, O, O, O, O, and O) (Sifi n).A small amount of bacterial mass from - suspicious colonies was transferred onto a slide and mixed each with one drop of test serum (ca. μl), so that a homogenous, slightly milky suspension developed.Th e results were read with the naked eye by holding the slide in front of a light source against a black background while swaying the slide (tilting it back and forth).To control for spontaneous agglutination, a negative control must be performed in parallel using physiological saline (NaCl) in place of the test serum.
From the colony which showed a positive reaction with polyspecifi c sera a subculture was prepared and strains were retested by using monospecifi c O:K antisera (Sifi n): O:K, O:K, O:K, O:K, O:K, O:K, O:K-, O:K-, O:K, O:K-, O:K-, O:K-, O:K-, O:K-, O:K, O:K, O:K, O:K-, O:K, O:K, O:K.A positive result had to be checked in the confi rmation test to exclude the eff ects of any parallel unspecifi c agglutination.Bacteria from the subculture was rinsed with NaCl and boiled for  hour to destroy the thermo-labile K-antigens.Th en the bacteria suspension was adjusted with NaCl to a germ density of Mc-Farland Standard  and used as an antigen suspension.

Results
Clinical stool specimens from  children with diarrhoea, their ages ranging from birth to <  years, were selected for the study over a period of  months (August  to December ).A study showed that / paediatric patients () had A variety of different serotypes of E. coli have been shown to cause diarrhoeal illness.The most common serotypes identified are shown in Table .
Among all serotypes of diarrhoeagenic E. coli the most prevalent in our study were O:K, O:K and O:K isolated from / patients (.).
The most prevalent EHEC strains found in this study were O:K-and O:K-.Two isolated strains of EIEC belong to serotype O:K-.Table .shows all isolated serotypes and their frequency.
Th e most prevalent serotypes of EPEC were O:K and O:K isolated from / patients (.),O:K from / patients (), followed by O:K-and O:K isolated from / patients (.).Among ETEC, the most prevalent serotypes were O:K found in / (.) and O:K in / patients (.) (Graphs . and .).

Discussion
Diarrhoea remains one of the main sources of morbidity and mortality in today's world and a large proportion is caused by diarrhoeagenic E. coli ().Th e frequency was investigated in many studies and ranges from -  (, , , ) with EPEC and ETEC as the most common (, , , , ,  and ).In our study,  of the patients had diarrhoea due to diarrhoeagenic E. coli.EPEC and ETEC were the most prevalent groups ( and . respectively).Th e most common serotypes were O:K, O:K, O:K (/ patients, i.e. . each) and O:K (/ patients, i.e. .).Th e percentage of cases of sporadic endemic infant diarrhoea which are due to ETEC usually varies from  to  (, ).Th e study of diarrhoeagenic E. coli in four diff erent age groups among  children in Th ailand () showed that the isolation rate of diarrhoeagenic E. coli was signifi cantly higher in children less than  year of age, compared with children older than  year.ETEC were more common in children older than  years, compared with the younger children.In our study, the average age of patients was  years, and ETEC was more common during the first twelve months.The reservoir of EPEC infection is thought to be symptomatic or asymptomatic children and asymptomatic adult carriers, including mothers and persons who handle infants ().Numerous studies have documented the spread of infection through hospitals, nurseries, and day care centres from an index case.In symptomatic patients, EPEC can be isolated from stools up to  weeks after cessation of symptoms In addition to profuse watery diarrhoea, vomiting and low-grade fever are common symptoms of EPEC infection.Faecal leukocytes are seen only occasionally ().
Th e most notable feature of the epidemiology of the disease due to EPEC is the striking age distribution seen in persons infected with this pathogen.EPEC infection is primarily a disease of infants younger than  years.As reviewed by Levine and Edelman numerous case-control studies in many countries have shown a strong correlation of isolation of EPEC from infants with diarrhoea compared to healthy infants ().Th e correlation is strongest with infants younger than  months.In children older than  years, EPEC can be isolated from healthy and sick individuals, but a statistically signifi cant correlation with disease is usually not found.In our study, EPEC has been isolated in all age groups (from  months to  years).
We have isolated EIEC from stool samples of two children aged  and  years.Epidemiologic studies of EIEC mostly describe outbreaks.In sporadic cases, many EIEC strains are probably misidentifi ed as Shigella spp.because EIEC strains are biochemically, genetically, and pathogenetically closely related to Shigella spp.().Documented EIEC outbreaks are usually foodborne or waterborne, although person-to-person transmission does occur ().
We report two cases of an E. coli O:K-infection in patients aged  years and  year with severe bloody diarrhoea and abdominal cramps.It progressed to haemolytic uremic syndrome (HUS) approximately  to  days after the onset of diarrhoea.Treatment was limited largely to supportive care.Th is syndrome is the most worrisome complication of EHEC infection because it is a serious and potentially fatal illness."Haemolytic" refers to the break-up of red blood cells which leads to anaemia.Th ere is also destruction of platelets (thrombocytopenia) which, in turn, promotes abnormal bleeding."Uremic" refers to the failure of the kidneys.In addition, problems in the brain with seizures and coma may occur.HUS most commonly aff ects children under the age of  years and is the most common cause of acute renal failure in infants and young children.A very low infectious dose for EHEC infection has been estimated from outbreak investigations.Th is number, on the order of  to  organisms for infection, is similar to the number required for Shigella infection and is consistent with the numerous reports of person-to-person transmission in outbreaks and in the institutional setting ().Th e incubation period of EHEC diarrhoea is usually  to  days, although incubation times as long as  to  days or as short as  to  days have been described in some outbreaks.Th e initial complaint is usually non-bloody diarrhoea, although this is preceded by abdominal cramps and pain, as well as a short-lived fever in many patients.Vomiting occurs in about half of the patients during the period of non-bloody diarrhoea and/or at other times during the illness.Within  or  days, the diarrhoea becomes bloody and the patient experiences increased abdominal pain.Th is stage usually lasts between  and  days.In severe cases, faecal specimens are described as "all blood and no stool" ().
This bacteria and the toxin produced (Shiga-like toxin) should be searched for in cultures of stools and colonic biopsies in the case of bloody diarrhoea, in particular when a haemolytic-uremic syndrome is associated.The isolation of E. coli O:H or other Stx-producing E. coli strains from stool specimens depends upon culturing early in the course of the disease.Unfortunately, many patients are not extensively evaluated until they have HUS symptoms, which usually begin several days after the onset of diarrhoea (, ).
Th e most common non-O:H serotypes associated with human disease include O:K, O:K, O:K, O:H.At least  outbreaks due to these organisms have been reported in Japan, Germany, Italy, Australia, the Czech Republic and the United States ().In our study, O:K-and O:K-were the most common serotypes of EHEC (. each).O:KO and O:K constituted  and , of all isolated DEC serotypes.
As with other diarrhoeal pathogens, the primary goal of treatment of diarrhoea due to DEC is to prevent dehydration by correcting fl uid and electrolyte imbalances.Oral rehydration may be sufficient for milder cases, but more severe cases require parenteral rehydration.Correction of nutritional imbalance with lactose-free formula or breast milk may be insufficient for some severely ill patients, and total parenteral nutrition may be required ().Th ere are no vaccines currently available or in clinical trials to prevent disease due to EPEC.
Although EHEC strains are generally susceptible to a variety of antibiotics, there are no prospective studies showing conclusively that the use of antibiotics alters the outcome of the disease.Th ere are, however, retrospective studies which suggest that patients who received antibiotics may be at greater risk of developing HUS.Th e use of antibiotics may be harmful for two potential reasons: fi rst, lyses of bacteria by some antibiotics leads to increased release of toxin, at least in vitro; second, antibiotic therapy could kill other intracolonic bacteria, thereby increasing the systemic absorption of toxin ().

Conclusion
Th is study confi rms that diarrhoeagenic E. coli is an important causal agent of diarrhoeal diseases in paediatric patients in Bosnia and Herzegovina.Enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are the most common.Th is is the fi rst report about the frequency and distribution of the most common serotypes of DEC in Bosnia and Herzegovina.Simultaneously, it is the fi rst report of E. coli O:H, an emergent pathogen causing the haemolytic-uremic syndrome, a serious and potentially fatal illness.Th erefore, research in diarrhoeagenic E. coli remains an important task to pursue.

Frequently
AMELA DEDEIĆLJUBOVIĆ ET AL.: FREQUENCY AND DISTRIBUTION OF DIARRHOEAGENIC ESCHERICHIA COLI STRAINS ISOLATED FROM PEDIATRIC PATIENTS WITH DIARRHOEA IN BOSNIA AND HERZEGOVINA Several recent studies have implicated DAEC strains as agents of diarrhoea, while other studies have not recovered DAEC strains more frequently from diarrhoeal patients than from asymptomatic controls.An age-dependent susceptibility may explain this observation, because when populations are stratifi ed by age, the association of DAEC with diarrhoea is found only in children older than infants.Th e reason for such an age-related phenomenon is as yet unknown.Other epidemiologic features, such as the mode of acquisition of DAEC infection, are also as yet undetermined

TABLE 2 .
Frequency of diarrhoeagenic E. coli serotypes and groups AMELA DEDEIĆLJUBOVIĆ ET AL.: FREQUENCY AND DISTRIBUTION OF DIARRHOEAGENIC ESCHERICHIA COLI STRAINS ISOLATED FROM PEDIATRIC PATIENTS WITH DIARRHOEA IN BOSNIA AND HERZEGOVINA During the fi rst twelve months, serotypes O:K and O:K of ETEC were the most common: / patients (.).Table . shows the age distribution of diarrhoeagenic E. coli.Th e average age of the patients was  years.