A Review on Pathogenic Escherichia coli in Malaysia

| Pathogenic / diarrheagenic Escherichia coli is a major foodborne pathogen worldwide, thus of great public health concern. These E. coli can be found in human, animals and environment, including soil and water. Infections caused by pathogenic E. coli may occur due to direct contact with infected animals and contaminated environment as well as consumption of contaminated or undercooked food and untreated water. This review highlights the occurrence of pathogenic E. coli in Malaysia.


IntroductIon
F oodborne illness is an unavoidable public health con- cern worldwide due to consumption of contaminated food ( Jianghong and Carl, 2007).Over 250 bacterial species are reported to cause foodborne illnesses in humans among which Escherichia coli (E.coli) is considered to be the cause of most of these illnesses (Carlos et al., 2003).Escherichia coli is commonly found in the intestinal tract of human which colonises in the gastrointestinal tract of infants within few hours after birth and thus, deemed as one of the first facultative organism to colonise the human gut (Nataro and Kaper, 1998;Fanaro et al., 2003).Maturation of the bacteria however, took several of years and typically confined to the lumen of gut and to the external layer of the intestinal mucous (Mansan-Almeida et al., 2013).E. coli is said to be highly versatile, colonizing wide range of mammals as well as birds (Beauchamp and Sofos, 2010).
Escherichia coli is divided in to two types, pathogenic E. coli and non-pathogenic E. coli.The non-pathogenic strains of E. coli described as commensal E. coli are present in the normal microflora of intestine which are harmless, hin-der the growth of harmful bacteria and produce vitamins (Nataro and Kaper, 1998;Beauchamp and Sofos, 2010).The pathogenic E. coli strains can be further classified into intestinal diarrheagenic E. coli which causes diarrhea and extraintestinal E. coli (ExPEC) which causes wide range of illnesses in humans such as the neonatal meningitis, chronic urinary tract infections, septicemia and hemolytic uremic syndrome (Nataro and Kaper, 1998;Chomvarin et al., 2005;Beauchamp and Sofos, 2010;Croxen and Finlay, 2010).
Enteroaggregative E.coli (EAEC) AAFs, cytotoxins EAEC strains have recently been identified as the second most frequent cause of travellers' diarrhoea associated with persistent diarrhoea in humans after ETEC in both developed and developing countries and recently acute diarrheal illness in newborns and children were observed (Croxen et al, 2010).Clinical features of EAEC include watery diarrhoea, sometimes accompany by bloody and mucus, with little to no vomiting along with low fever.
Diffusely Adherent E. coli (DAEC) Daa, AIDA DAEC usually infects children under the age of 1 to 5 years (Levine and Edelman, 1984;Nataro and Kaper, 1998).There were few clinical cases for study and most of DAEC patients recorded fecal leukocytes or watery diarrhoea without blood (Poitrineau et al., 1995).
Adherent invasive E. coli (AIEC) Type 1 fimbriae, cellular invasion Adherent invasive E. coli (AIEC) has been considered as one of the most important causative agent for Crohn's disease (CD), which when affect the small bowel cause inflammation and is known as inflammatory bowel disease (IBD).Unlike other pathogenic E. coli strains, AIEC pathotype does not express common virulence factors.Therefore, the invasive phenotype and its proinflammatory genetics is not fully understood (Nash et al., 2010).
Of all diarrheagenic E. coli identified, Shiga-toxin or Vero toxin producing (STEC/VTEC) EHEC is the most important pathotype in human diseases (Wani et al., 2003).
There are many serotypes in STEC and among them, the EHEC serotype O157:H7 is found to be highly virulent, responsible for causing outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) around the globe.Ruminants are recognized as natural reservoir hosts for E. coli O157:H7 (Nataro and Kaper, 1998).No treatment has yet been found for the infections caused by EHEC (Goldwater and Bettelheim, 2012).The non-availability of treatment of EHEC imparts more attention towards the study of epidemiology, pathology and control measures in case of outbreak.
Pathogenic E. coli can be found in contaminated environment (water and soil) because they are being shed in the faeces of infected animals and humans.Contamination of animal products may be due to inappropriate practices during slaughtering and dressing process, especially from intestinal contents and faeces during evisceration (Bhunia, 2007).

ePidemiology of PathogeniC E. coli
The epidemiology of each pathogenic E. coli was reported to vary according to different species and strains of E. coli.
The presence of these pathogenic E. coli was found in various animal reservoirs and spread within and as well as to other animals (Croxen et al., 2013).Numerous epidemiology studies carried out found that various factors contribute to the shift of prevalence based on different geographical areas, population, age distribution, socioeconomic class and detection methods (Ochoa et al., 2008).
The epidemiology of EHEC has been of a major focus and deem important among the researchers, especially on the detection of EHEC serotype, O157:H7, although other non-O157 strains are also major causes of many outbreaks in many regions including North America, Australia and Europe (Allos et al., 2004;Angulo, 2007) (Rivero et al., 2010).On the other hand, the neighbouring country, Brazil, showed low incidence of HUS and cases of O157:H7 are rare (Irino et al., 2002).This may be associated with known risk factors such as contaminated meat consumption, playing in contaminated recreational water and poor personal hygiene (Bentancor et al., 2011).Although EHEC infections are detected in many developing countries, the widespread of EHEC still remain unclear due to lack of surveillance and clinical diagnosis especially in the sub-Saharan regions.
In Kenya, case studies by GEMS (Global Enteric Multicenter Study) indicated that EPEC significantly causes moderate to severe diarrhoea in children under the age of 2 years.Continuous studies later revealed that EPEC infections was not strongly related to causing moderate to severe diarrhoea, however, if present, it may increased the risk of death among newly born to 11 months old babies (Kotloff et al., 2013).In another case occurred in the United States, only 4 patients were reported due to EPEC infections (CDC, 2013).It was concluded that the occurrence of EPEC decreases with increase of age and that infections in adults are extremely rare (Nataro and Kaper, 1998).This phenomenon perhaps was associated to the loss of certain EPEC receptors with age or development of the immunity in humans (Nataro and Kaper, 1998).Previously, EPEC was thought to occur predominantly in industrialized countries, however, new findings indicate that EPEC was also reported in developing countries (Afset et al., 2003;Alikhani et al, 2006;Nguyen et al., 2006;Bakhshi et al., 2013).Although, in many countries, EPEC is consider as less important cause of diarrhoea, nonetheless, EPEC still poses serious health concern to children aged 2 and below and the epidemic of this pathogen may well persist in other parts of the world where such epidemiology studies had never been carried out and that the possibility of reemerged of this infection cannot be ruled out.
The outbreak of EIEC has always been under represent- ed in epidemiology studies due to its less pathogenicity towards human as compared to other pathogenic E. coli pathotypes.Furthermore, due to its genetic, pathogenic and biochemical similarities to Shigella, it is always being misdiagnosed.A comprehensive overall picture on the epidemiology of EIEC is possible only with molecular detection method, targeting specific EIEC gene markers (Croxen et al., 2013).Thus, in the last decade, only a handful of cases was reported in Central and South America, Africa and Asia (Ratchtrachenchai et al., 2004;Okeke, 2009;Perez et al., 2010).The largest outbreak of EIEC was reported in 1985 which affected 370 people in Texas, United States (Gordillo et al., 1992).The epidemiology of Shigella was far more documented and is reported to associate with about 30% to 50% of bacillary dysentery cases worldwide (Pfeiffer et al., 2012).

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Due to the limited surveillance implementation globally for all pathogenic E. coli pathogtypes, information on the incidence of EAEC is limited to certain parts of the world such as North and South America and Europe (Nataro et al., 2006).A large scale study carried out in the United States, revealed that EAEC was the most commonly found bacteria in the emergency departments and outpatients clinics of two large academic hospitals (Nataro et al., 2006).A significant large outbreak of EAEC was recorded almost a decade ago, in 1997, which occurred in a school lunch and affected 2,697 school children in Japan (Wanke et al., 1991).In that same year, approximately 15% of a village population in India was affected by EAEC.Subsequently, diarrheagenic EAEC cases was also reported in Mali (Boisen et al., 2011), Libya (Dow et al., 2006), sub-Saharan Africa (Kotloff et al., 2013) and Nigeria (Okeke et al., 2010).Over 900 patients developed HUS and later were found to be infected with hybrid pathogens which carried virulence genes from both EAEC and STEC (Mellmann et al., 2011;Estrada-Garcia and Navarro-Garcia, 2012).
Although ETEC is the primary cause of traveller's diarrhoea, cohort studies in Bangladesh, Argentina, Egypt and Guinea-Bissau, showed that ETEC is significantly associated to morbidity and mortality in children in developing countries (Viboud et al., 1999).Approximately 280 million of children aged 0 to 4 years were reported to experience ETEC induced diarrhoea (Wenneras and Erling, 2004).In many developed countries, diagnosis for ETEC is uncommon for patients presented with diarrhoea.However, it is the most common bacterial related to traveller's diarrhoea, constituting approximately 30% of such cases (Shah et al., 2009).The largest outbreak of ETEC occurred in 1998, in the state of Illinois, Unites States where approximately 3,300 people were believed to have become ill through consumptions of foods prepared by infected personnel (Beatty et al., 2006).Subsequent outbreaks were also reported in Nevada ( Jain et al., 2008) and Illinois (Yoder et al., 2006) from a sushi restaurant and a buffet style lunch respectively.Such outbreaks have also occurred in other developed countries such as in Denmark (Ethelberg et al., 2010), Japan (Konishi et al., 2011) and Israel (Huerta et al, 2000), in which the sources of infection were associated to contaminated water and foods.
The detection for DAEC strain still remains undeveloped and to date, there is no universal protocol for detection of DAEC in clinical laboratory, Hence, the epidemiology and occurrence pattern of DAEC is unclear.However, DAEC isolates were discovered from children with diarrhoea in many countries such as Chile (Levine et al., 1993), Mexico (Giron et al., 1991), Australia (Gunzburg et al., 1993) and the United Kingdom (Knutton et al., 2001).In Brazil, 23% of 1,801 E. coli isolates from 200 children with diarrhoea were identified as DAEC which suggest that it may be more widespread than previously thought (Gomes et al., 1998).One interesting finding from above mentioned studies revealed that DAEC was also identified in healthy individual of the same age control group, suggesting that DAEC may also present in health people without diarrhoea.Thus, there is an urgent need to develop detection methods in clinical setting to specifically differentiate and identified DAED strains accurately and also to discover its mode of transmission as well as responsible reservoir hosts.There are also limited data on the epidemiology of AIEC, however, it was suggested that AIEC is correlated with Crohn's disease (CD), which was evident in several clinical studies that found AIEC isolates in CD patients (Darfeuille-Michaud et al., 2004;Martin et al., 2004).
With that, it is essential to carry out more clinical studies to further understand the transmission dynamics of AIEC globally, especially regarding its roles and connections with CD patients.

transmission of PathogeniC E. coli
Animal faeces are considered to be the major source of pathogenic E. coli.The close contact among animals in the farms may lead to the transmission to other animals.(Karch et al., 2005).Animal wastes, sewages from farming operations, manure/slurries which are frequently used as fertilizers for the crops or silage preparation and cattle grazing also contribute to the infection and re-infection of cattle.( Jiang et al., 2002;Kudva et al., 1997).
The presence and sustainability of Shiga toxin-producing E. coli (STEC) in the soil favours its infections in cattle and their presence in the environment also pose risk for human infections (Gagliardi et al., 2002;Howie et al., 2003;Ogden et al., 2002).
Pathogenic E. coli may be found in carcasses meat of infected animals or contaminated by faeces of infected an- imal through improper slaughtering and processing, and the unhygienic practices of workers in the farm can lead the pathogen to contaminate the milk during milking.Ingestion of raw or uncooked contaminated beef, drinking unpasteurized milk or untreated water can lead to infection by pathogenic E. coli, especially serotype O157 (Karch et al., 2005).

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EPEC can be transferred through contaminated foods such as vegetables, cheese, tuna fish, potato, macaroni salads, untreated water and through toys, rubber nipples and fomites among children.EAEC can be transmitted through food.The method of transmission of DAEC is not yet identified ( Jianghong and Carl, 2007).
Transmission of pathogenic E. coli infection from person to person may occur because of unhygienic measures.It can spread within community like families and close contacts through oral route especially among children (Karch, et al., 2005).An outbreak was reported to be caused by transmission of the disease from patients to their family members (Parry, 1998).
Many cases in children infected with pathogenic E. coli were due to their contact with the infected animals in the farms, petting zoos or environment contaminated with animal faeces.The infections can occur in persons and children who do not wash their hands (CDC, 2009).
Government regulatory agencies, environmentalists, beach managers and sewage operators are concern over the presence of E. coli.Proper risk assessment and control procedures are to be adopted for water control, as E. coli in water can originate from human and nonhuman sources, such as farms, wild animals, waterfowl, and pets (Harwood et al., 2000;Krumperman, 1983;Parveen et al., 1997).

isolation of E. coli o157
For qualitative analysis of E. coli O157, direct plating is frequently practiced which later on is improved by using immunomagnetic separation (IMS) technique.According to Šafařiková and Šafařik (2001), the IMS technique performed more sensitive detection of specific microorganism in comparison with direct plating.Small portion of samples can also be sensitively identified on selective media such as Sorbitol MacConkey with Cefixime Tollurite (CT-SMAC), followed by IMS (LeJeune et al., 2006;Khanjar and Alwan, 2014;Dodd et al., 2003;Chapman et al., 1994;LeJeune et al., 2004;Omisakin et al., 2003).
The PCR is a sensitive technique for the characterization of different isolates and it can also differentiate pathogenic from non-pathogenic E. coli.The pathotypes of E. coli can be differentiated by the PCR based upon the existence of virulence genes available in each pathotype.Specific set of primers in the PCR can be used to amplify the virulent genes (Kalnauwakul et al., 2007).PCR has been used commonly for the epidemiological investigation of pathogenic E. coli infections worldwide (Olsvik et al., 1991).A number of studies showed the use of multiplex PCR (m-PCR) for the detection of pathogenic E. coli (Wani et al., 2003;Wani et al., 2005;Kalnanwakul et al., 2007;Fagan et al., 1999).Watterworth, (2005) was able to design an m-PCR by using six sets of specific primers for the detection of four different pathotypes of pathogenic E. coli which were lt and st for ETEC, eaf for EPEC, stx 1 and stx 2 for STEC and ial for EIEC.Multiplex PCR is also able to differentiate between pathogenic E. coli and other enteric bacteria.Osek, (2001) also designed an m-PCR to differentiate between the ETEC pathotype and other gram negative bacteria using specific primers for the detection of heat stable (st) and heat labile (lt) genes of the ETEC pathotype.Chang et al., (2013) targeted rfbO157 and fliCH7 for the detection of E. coli O157:H7.

oCCurrenCe of PathogeniC E. coli WorldWide
The occurrence of pathogenic E. coli is worldwide is tabulated in Table 2.

oCCurrenCe of PathogeniC E. coli in malaysia
Among the pathogenic E. coli, the EHEC serotype O157:H7 is of utmost importance due to its serious implications in humans and the increasing reported occurrence in many regions around the globe (Willshaw et al., 1994;Dundas et al., 2001;Effler et al., 2001) specifically in United States and Japan (Rangel et al., 2005;Muto et al., 2008).Thus, most studies on E. coli carried in Malaysia, focused mainly on the serotype O157:H7.In Malaysia, the food-borne bacteria such as Salmonella, Listeria, Staphylococcus, Campylobacter and E. coli, were isolated from animal and animal products (Adzitey et al., 2012;Saleha, 2002;Arumugaswamy et al., 1995).
Table 3 shows some studies conducted on prevalence of E. coli and its pathogenic strains among different samples in Malaysia.

table 2 :
Nanu et al. (2007)ogenic E. coli worldwide of barley and corn fed cattle were examined by both IMS and direct plating techniques for the detection of E. coli O157.7.4% (42) were positive for E. coli O157 by IMS while 3.3% (19) were positive for E. coli O157 using the direct plating procedure.225samplescollected from hides of barley-fed cattle, only 3 (1.33%)werepositive for E. coli O157:H7 while only 1 (0.44%) of 225 samples collected from the corn fed cattle was positive for E. coli O157:H7.collectedincludingfaeces, hides and carcasses, 20 in each of three separate trial periods.E. coli ranged from 98 to 55%.Highest E. coli presence was recorded from at preevisceration carcass samples at 40.4%.Nanu et al. (2007)India 240 raw milk samples were collected from three (3) farmer societies.E. coli was detected in 31.6%(76) of raw milk samples while Staphylococcus aureus at 35% (84).Most of the E. coli isolates were consisted of serotypes 05, 024, 025, 068, 084, 087, 0103, 0116, 0125, 0145, 0157 and 0172., 100 each of wild birds including quails, doves, sparrows and cattle egrets, and 150 stool samples of diarrheic and non-diarrheic humans, were collected.E. coli was isolated at 48% while Salmonellae at 10.75%.The individual prevalence of E. coli among quails was 47%, doves 49%, sparrows 13.2% and cattle egrets 43.6% while E. coli was detected among 56% in humans stool samples.The antibiogram study revealed that the isolates were 100% sensitive to tetracycline and gentamicin, which is the drug of choice for the treatment of diarrheagenic E. coli in calves.
environmental factors and workers personal hygiene play an important role in microbial contaminations.This study may serve as a template to investigate the role of human, animal and environmental factors in contamination of E. coli and other microbes relevant to food safety.Advances in Animal andVeterinary Sciences February 2018 | Volume 6 | Issue 2 | Page 100

Advances in Animal and Veterinary Sciences February 2018 | Volume 6 | Issue 2 | Page 101table 3 :
Prevalence of E. coli and its pathogenic strains among different samples in Malaysia.Escherichia coli O157:H7 were detected in tenderloin beef and chicken meat burger respectively.All the strains carried XbaI genes.All the isolates showed resistance to one or more than three antibiotics among the 14 antibiotics tested.