Escherichia coli flora and diarrhea in Nicaraguan children

A combination of a phenotyping method (PhP typing) for the identification of clonal groups, and a genotyping method (PCR) for the identification of virulent strains of E. coli were used in order to obtain insight into diarrheal disease morbidity and epidemiology of diarrheagenic E. coli strains in children in León, Nicaragua. Fecal samples from 381 children aged 0-5 years suffering from diarrheal disease and 145 healthy children of the same age were analyzed. Besides, a cohort of 20 healthy infants aged 0-12 months were followed by repeated sampling (8 samples per infant). All samples were analyzed by a multiplex PCR (EAEC, ETEC; EPEC, EHEC, EIEC markers) on the primary streak and from the 282 positive samples 2,164 E. coli isolates were analyzed by single-colony PCR, and from all samples, eight E. coli isolates were phenotyped; totally 4,753 E. coli isolates were analyzed. Our main results are: Diarrheagenic E. coli (DEC) positive samples were found at high rates in infants aged 3- 60 months (Paper I), but were rare in infants below 3 months of age (Paper IV). Thus, colonization by DECs seem to start early in life and increase with age. The phenotypic diversities among all isolates from diarrheal cases and from healthy children were equal and high, thus giving indication that no large outbreak of DEC diarrhea occurred during the time period studied (Paper II) EAEC and EPEC could not be correlated to diarrhea, whereas ETEC and EHEC were significantly more common in diarrheal cases (Papers I, II, and III). ETEC estA and EHEC were only found in diarrheal cases, and isolates positive for these DEC types belonged to a few PhP types that could represent pathogenic clones (Paper III) DEC positive isolates seldom dominated the fecal E. coli flora, but for all DEC types, except for EAEC, a high proportion of DEC positive isolates in the fecal E. coli flora seemed to be correlated to diarrhea. Thus, a low proportion of DEC in the fecal E. coli flora may not be of significance for development of diarrhea, but may represent occasional carriage of virulence gene(s) by normal flora strains (Paper III). Newborns were found to rapidly exchange their E. coli strains during their first year of life. Colonization by single DEC positive isolates was rarely correlated to diarrheal disease (Paper IV). Our findings suggest that there are certain stable types or clones of diarrheagenic E. coli that can circulate in a population, and have the possibility to colonize the intestine and to dominate the E. coli flora, thereby causing diarrhea. These are the true DECs. However, the virulence genes are promiscuous and may spread to the E. coli bacteria of the normal flora, whereupon these scarce gene markers are detected by the sensitive PCR test in samples from both healthy and diseased individuals, without necessarily having a pathogenic role. Thus, the obtained information on the distribution of DEC pathotypes and their diversities may be used to optimize the use of today s diagnostic tools, which in turn might affect treatment approaches and vaccine development strategies

One Health-One City; the extent of Shiga-toxin producing Escherichia coli in Cape Town

The estimated global burden of STEC (Shiga toxin producing Escherichia coli) is 2,481,511 illnesses, 269 deaths, and 26,827 DALYs with 48% of these being foodborne. This thesis provides information on STEC diagnostic strategy, undetected STEC in a tertiary referral hospital in Cape Town, and the virulence and antimicrobial resistance properties of tellurite resistant diarrheic E. coli isolated on CHROMagar(TM)STEC (CHROMagar Microbiology, Paris, France). Deploying the One - Health surveillance approach to study selected diarrheic bacterial pathogens in an informal settlement setting, this study sheds light on the extent of bacterial foodborne pathogens in human and non-human sources

Assessment of the incidence of E.coli in Tyume and Buffalo rivers in the Eastern Cape Province of South Africa

Waterborne diseases are among the leading causes of morbidity and mortality in developing countries and every year around 2.2 million people die due to basic hygiene related diseases like coliform diarrhoea. Universal access to safe drinking water and sanitation has been promoted as an essential step in reducing these preventable diseases (Tambekar and Banginwar, 2005; Patil, 2004; Charan, 2004). Diarrheagenic Escherichia coli are one of the most important etiologic agents of acute diarrhea and represent a major public health problem in developing countries like South Africa The present study was conducted between August 2010 and July 2011 to investigate the prevalence and distribution of virulent E. coli strains from water samples collected from Tyume and Buffalo River, located in Eastern Cape Province of South Africa using conventional microbiological methods and PCR analysis. Twelve Water samples were collected from three different sites of the rivers, viz; upstream, middle stream and the downstream of the dam. E.coli was isolated by the membrane filtration method on mFC. A total of 374 isolates from both rivers were identified by convenctional microbiological techniques. For both Buffalo and Tyume River, A large proportion (87 and 114, respectively) of the isolates from the mid stream samples satisfied the identification characteristics for E. coli (blue colonies on MFC agar and violet/purple colonies on Chromocult agar) and thus revealing high levels contamination when compared to isolates from the downstream (55 and 47) and the upstream (30 and 31) All the isolates that satisfied the primary identification stage were subjected to PCR. DNA was extracted using the boiling method and then the DNA was used as a template for PCR. Specific PCR analysis was performed on all E. coli isolates to amplify the alr gene that codes for alanine racemase Out and of the 202 isolates amplified for Tyume river, 70 (35 percent) were positively identified as E. coli since they possessed the alr gene fragment. and out of the 172 isolates amplified from Buffalo River, 80(47 percent) were also positively identified as E. coli. For both Tyume and Buffalo River, the highest prevalence was observed midstream (39 percent and 56 pecent respectively). The identified E. coli were further characterized into different pathotypes. Amplification of the shig gene, LT gene, EaeA gene, Eagg gene and the ST gene were used to detect pathogenic E.coli. In Tyume River, Genes of ETEC (lt or st) were detected in 21/70 (30 percent); Gene of EPEC (eae) was detected in 14/70 specimens (35 percent); Genes of EAEC (Eagg) was detected in 14/70(35 percent) and genes of EIEC (shig) were detected in 11/70(16 percent). In Buffalo River, no DEC was recovered upstream and downstream of the river. EAEC (8 percent) was the only pathotypes recovered midstream of the river. Strains of all five E. coli categories showed high-level resistance to ampicillin, tetracycline, cotrimoxazole, and chloramphenicol but were highly susceptible to quinolones, aminoglycosides, and novobiocin. The highest resistance (100 percent) amongst the isolates was observed to ampicillin by EAEC, Heat Labile (ETEC) and EIEC, followed by 87.5 percent by EAEC to carbenicillin. The highest susceptibility was to quinolones (100 percent) by all the four categories of E.coli. The screening for antibiotic resistance genes revealed the absence of SHV, CTMX and TetC genes as they were not detected in any of the E.coli isolates. However, TEM genes were observed in 80 percent of the isolates. Integron conserved segment was detected in these same organisms in the same proportion as TE

Characteristics and Etiology of Moderate-to-Severe Diarrhea of Acute, Prolonged Acute, and Persistent Duration among Children Less than 5 Years Old in Rural Western Kenya, 2008-2010

Worldwide, diarrheal disease is the second leading cause of death in children under 5 years old. Data on diarrhea of extended duration is limited. We described the characteristics associated with acute, prolonged acute and persistent diarrhea in Kenyan children less than 5 years of age participating in the Global Enterics Multicenter Study. Children presenting at a clinic were enrolled if they met the case definition for acute moderate-to-severe diarrhea defined as \u3e3 loose stools in the last 24 hrs, within 7 days of illness onset, with \u3e1 of the following: sunken eyes, skin tenting, dysentery, IV rehydration, or hospitalization. To determine diarrhea duration, the child’s caretaker was asked to recall the number of days the child had diarrhea in the 7 days pre-enrollment, and to record each day of diarrhea post-enrollment on a form for 14 days. Stool specimens were collected at enrollment, and the post-enrollment form was collected during a home visit. We defined acute diarrhea (AD) as \u3c6 days\u3eduration, prolonged acute diarrhea (ProD) as 7-13 days, and persistent diarrhea (PD) as \u3e14 days. From January 31, 2008 to January 24, 2010, 557 children with acute moderate-to-severe diarrhea were enrolled. Using the Wilcoxon rank-sum test, Kruskal-Wallis test, and Cox Proportional Hazards Model we examined the relationship between the duration of diarrhea by gender, age, and various etiologic agents. We found no association between gender and the duration of diarrhea. Age was associated with diarrhea of extended duration; children less than or equal to 11 months of age were 1.3 times more likely to experience diarrhea of longer duration than their counterparts. We found Cryptosporidium to be more associated with ProAD and PD. Children infected with Cryptosporidium were 1.5 to 1.7 times more likely to have diarrhea with a longer duration than their counterparts. Based on these results, interventions related to diarrhea and diarrhea of extended duration should focus more closely on young children, especially children less than 24 months of age

Pathogenicity assessment of Shiga toxin‐producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC

The provisional molecular approach, proposed by EFSA in 2013, for the pathogenicity assessment of Shiga toxin‐producing Escherichia coli (STEC) has been reviewed. Analysis of the confirmed reported human STEC infections in the EU/EEA (2012–2017) demonstrated that isolates positive for any of the reported Shiga toxin (Stx) subtypes (and encoding stx gene subtypes) may be associated with severe illness (defined as bloody diarrhoea (BD), haemolytic uraemic syndrome (HUS) and/or hospitalisation). Although strains positive for stx2a gene showed the highest rates, strains with all other stx subtypes, or combinations thereof, were also associated with at least one human case with a severe clinical outcome. Serogroup cannot be used as a predictor of clinical outcome and the presence of the intimin gene (eae) is not essential for severe illness. These findings are supported by the published literature, a review of which suggested there was no single or combination of virulence markers associated exclusively with severe illness. Based on available evidence, it was concluded that all STEC strains are pathogenic in humans, capable of causing at least diarrhoea and that all STEC subtypes may be associated with severe illness. Source attribution analysis, based on ‘strong evidence’ outbreak data in the EU/EEA (2012–2017), suggests that ‘bovine meat and products thereof’, ‘milk and dairy products’, ‘tap water including well water’ and ‘vegetables, fruit and products thereof’ are the main sources of STEC infections in the EU/EEA, but a ranking between these categories cannot be made as the data are insufficient. Other food commodities are also potentially associated with STEC infections but rank lower. Data gaps are identified, and are primarily caused by the lack of harmonisation in sampling strategies, sampling methods, detection and characterisation methods, data collation and reporting within the EU.info:eu-repo/semantics/publishedVersio

Molecular Approaches in the Diagnosis of Diarrhoeal Disease in Children from Rural Gambia

Enteroaggregative Escherichia coli (EAEC) is a genetically diverse enteric pathogen that causes growth faltering among children, acute and chronic diarrhoea among children and adults living in both industrialised and low income countries. The German outbreak of EAEC-Shiga-toxin-E. coli in 2011 resulted in over 4000 confirmed cases of diarrhoea with over 54 fatalities in 14 European countries as well as United State of America and Canada. Several studies conducted in sub-Sahara Africa (SSA), Latin America and Asia countries have identified EAEC more frequently than any other bacterial pathogens. In SSA, case fatality due to EAEC is not well documented but the morbidity rate particularly among younger children is huge. Studies conducted in Senegal, Central-Africa-Republic and Tanzania showed that EAEC were endemic among HIV-positive patients with diarrhoea. Few studies from SSA have reported distribution of antimicrobial resistance pattern of EAEC. The Global Enteric Multisite Study (GEMS), a three-year case-control study conducted in seven African and Asia countries, showed that the prevalence of EAEC was higher among children with no diarrhoea (463/741, 62.5%) compared to children with diarrhoea (278/741, 37.5%). The aim of this retrospective analytical study nested to GEMS is to explore other molecular approaches that identify infectious EAEC and to show the genetic diversity and antimicrobial resistant pattern of EAEC. Study design of the first approach involves unmatched case-control 428 (157 cases and 271 controls) EAEC isolates that were examined by polymerase chain reaction (PCR) for the presence of 21 common EAEC virulence genes. This investigation implicated plasmid-encoded toxin (pet), AAF/1 fimbrial subunit (aggA) and hexosyltransferase homolog (capU) to be associated with diarrhoea in infants. In addition, two other virulence genes; Shigella exracellular protease A (sepA) and EAEC-heat-stable enterotoxin 1 (EAST1) were implicated in the EAEC that cause diarrhoea among children under 5 years old. The second approach utilised qualitative PCR (TaqMan-qPCR) method to assess the use of bacterial load diagnostic tool to diagnose infectious EAEC on selected matched case-control 160 (80 cases and 80 controls) EAEC isolates. Two biomarker genes, aatA and aaiC were the target in this study and both resulted in higher rate of higher bacterial load in controls (58/80 [72.5%]) compared to cases 48/80 [60%]), p – value 0.096. The third approach explored bacterial biofilm formation to diagnose infectious EAEC on 400 unmatched cases (150) and controls (250) EAEC isolates. Infectious EAEC produces biofilm to consolidate its colonisation in the host and damage to the tissue. The result of this study showed higher proportion of biofilm-producing EAEC in controls (61%) compared to cases (39%). However, biofilm-producing EAEC isolates that has aggR gene combined with one or all of the following virulence genes aatA, Aap, Orf3 and Orf61 revealed strong association with diarrhoea. Investigation into the antimicrobial resistant EAEC on the same 400 unmatched EAEC isolates revealed multi-drug resistant (MDR) EAEC infection as a significant problem among infants in the Gambia. MDR EAEC strains are almost equally distributed among cases and controls, and high (>71%) rate of resistant to Ampicillin, Sulphamethoxazole-trimethoprim and Tetracycline, and moderate (25%) rate of resistant to Chloramphenicol among study children. However, over ninety-four percent of the Gambia EAEC strains are susceptible to Cefotaxime, Ceftazidime, Ceftriaxone, Ciprofloxacin, Gentamicin and Amoxicillin-clavulanic acid. Additionally, result of whole genome sequencing (WGS) on 50 randomly selected EAEC isolates showed average 94% concordance of resistance genes with phenotypic disc diffusion method. This thesis provides detailed initial description and exploration of virulence genes associated with EAEC strains circulating in the rural Gambia and has revealed the likely biomarker genes to target in the diagnosis of infectious EAEC that cause diarrhoea in infant

Isolation and characterization of E. coli and Campylobacter spp. from diarrhoeal samples collected from selected hospitals in Amathole District Municipality, Eastern Cape, South Africa

Approximately 2-4 billion cases of infectious diarrhoea occur every year, with the highest numbers recorded in sub-Saharan Africa. It remains the most common public health issue among children in developing nations. The purpose of this research was to unfold the prevalence of diarrhoeagenic E. coli and Campylobacter pathotypes as well as elucidate their antibiogram characteristics in diarrhoeal stool samples collected in some medical facilities in Eastern Cape Province, South Africa. Two hundred stool samples were collected from both inpatients and outpatients from male and females of all age groups attending selected medical facilities in the study area. Isolation and characterization of both organisms were done using culture based and molecular methods. Antibiotic susceptibility patterns of identified isolates were determined against a panel of 12 antimicrobial agents. One hundred and twenty presumptive E. coli isolates and 42 presumptive isolates of Campylobacter spp. Were isolated. Eighty-two percent (82 percent) of the presumptive E. coli isolates were confirmed as E. coli while 46.3 percent belonged to Campylobacter spp. Pathotyping of the diarrhoeagenic E. coli isolates by Polymerase chain reaction (PCR) showed the following prevalences: DAEC 43 (32 percent), EHEC 18 (17 percent), EIEC 11 (10 percent) and EPEC 18 (17 percent). EAEC and ETEC were not detected, while for Campylobacter spp. 37 (88 percent) were C. jejuni, and C. coli was not detected. A total of 12 (32.4 percent) of the confirmed Campylobacter jejuni isolates were found to possess the fliM gene, 9 (24.3 percent) possessed the flhA gene and only 6 (16.2 percent) harboured the gene flgE2. None were positive for the flaA, flab and flhB genes.The antibiotic resistance patterns observed among the E. coli isolates were high against ampicillin (98.1 percent), chloramphenicol (94.3 percent) and tetracycline (90.6 percent). For Campylobacter spp., resistance observed were: chloramphenicol (91.6 percent), tetracycline (25.2 percent), erythromycin (49.6 percent) and gentamycin (56.4 percent). A lesser resistance against imipenem (35.9 percent) and quinolone (ciprofloxacin) (45.5 percent) were exhibited by the E.coli isolates. 10.8 percent and 20.3 percent of the Campylobacter isolates were resistant to imipenem and ciprofloxacin respectively. The presence of chloramphenicol (CatA1) and tetracycline (tetA) resistance genes were detected in 94 percent and 89 percent of E. coli isolates respectively while 98 percent of Campylobacter spp. Harboured the catA1 resistance gene. It could be deduced from this study that E. coli and Campylobacter spp. are predomiant enteric pathogens as the etiologic agents of diarrhoea in the study community, and that their antimicrobial resistance is high in the study location. The need to develop strategies to prevent infection and control resistant organisms is evident

The pathogenicity of enteroaggregative <i>Escherichia coli</i>

Strains of enteroaggregative Escherichia coli (EAggEC), characterised by their pattern of adhesion to HEp-2 cells known as the `stacked brick' formation, are a significant cause of chronic diarrhoea in certain under-developed countries. Strains of EAggEC are detected either by a HEp-2 adhesion cell test or by an `aggregative adhesion' gene probe. The pathogenic mechanisms expressed by EAggEC are only poorly understood and the aim of the research described was to obtain a better understanding of how these bacteria cause disease. The adhesion of EAggEC to HEp-2 cells was shown in the majority of strains not to involve fimbriae and was thought to result from physical properties of strains such as charge, since EAggEC adhered to `fixed' HEp-2 cells and readily agglutinated a range of different erythrocytes. Certain strains of EAggEC, which also hybridised with a probe for diffuse adhesion, expressed membrane-associated proteins (MAPs) of 18 or 20 kDa responsible for HEp-2 adhesion. Divalent cations were essential for the expression of the MAPs, which did not contain disulphide bonds or have a quaternary structure. Strains of EAggEC did not express recognised subunit toxins such as Verocytotoxin or E. coli heat-labile toxin, and strains which hybridised with probes for enteroaggregativeh eat-stable toxin-1 did not produce E. coli heat-stable toxin detected by the infant mouse test. Some EAggEC strains (15%) had haemolytic properties. Certain strains expressed type II capsular polysaccharides and approximately 50% of strains expressed an aerobactin-mediated iron uptake system. It was concluded that strains of EAggEC belonged to a very diverse range of serotypes, and it was thought that this heterogeneity resulted from strains of E. coil readily acquiring the genes encoding the EAggEC phenotype. Strains of EAggEC were not associated with a single pathogenic phenotype and the ability of these bacteria to adhere to HEp-2 cells in a `stacked brick'-pattern remains the only common characteristic

Evaluation of the incidence of enteric viruses, Vibrio species and Escherichia coli pathotypes in effluents of two wastewater treatment plants located in Keiskammahoek and Stutterheim in the Eastern Cape Province of South Africa

South Africa is currently experiencing water shortage crisis, a challenge that has been attributed not only to the scarcity of freshwater, but also to fast degrading water quality. Factors such as rapid urbanisation, population and economic growth, climate change as well as poor operational and maintenance of many of the exisiting water/wastewater treatment facilities have been acknowledged as important contributors to degrading water quality in the country. Untreated or inadequately treated discharged wastewater effluents constitute point source pollution to many freshwater environments in South Africa. Hence, it becomes imperative to evaluate wastewater discharges in other to protect the scarce freshwater resource, the environment and public health. Over a twelve-month sampling period (September 2012 to August 2013), we assessed the bacteriological, virological and physicochemical qualities of the discharged final effluents of two wastewater treatment facilities in the Eastern Cape Province of South Africa. For the physicochemical assessment, a total of 144 final effluent samples were collected from both the final effluent tanks (FE) and the discharge points (DP) of the treatment facilities. Physicochemical parameters including pH, temperature, turbidity, total dissolved solids (TDS), dissolved oxygen (DO), electrical conductivity (EC) and free chlorine concentration were determined on site while biological oxygen demand (BOD), nitrate (NO3-), nitrite (NO2-), phosphate (PO4-) and chemical oxygen demand (COD) were determined in the laboratory. The bacteriological analysis of the samples was done using standard membrane filtration (MF) technique. Bacterial group assessed included: faecal indicator bacteria (faecal coliforms and E. coli) and Vibrio species, while the antibiotic susceptibility profiles of selected E. coli and Vibrio species isolates against some selected antibiotics commonly used in human therapy and veterinary medicine were determind using the standard agar-disc diffusion method. The occurrence and concentrations of human enteric viruses including: human adenovirus (HAdV), hepatitis A virus (HAV) and rotavirus (RoV) in the samples were determined by TaqMan-based real-time polymerase chain reaction (qPCR) following concentration by adsorption-elution method. The physicochemical characteristics of the samples ranged as follows: pH (6.5 – 7.6), TDS (95 – 171 mg/L), EC (134 – 267 μS/cm), temperature (12 – 27 °C), turbidity (1.5 – 65.7 mg/L), free chlorine (0.08 – 0.72 mg/L), DO (2.06 – 9.81 mg/L), BOD (0.13 – 9.81 mg/L), NO3- (0 – 21.5 mg/L), NO2- (0 – 0.72 mg/L), PO4- (0 – 18.3 mg/L) and COD (27 – 680 mg/L). Some of the characteristic such as pH, TDS, EC, temperature, nitrite and DO (on most instances) complied with recommended guidelines. Other characteristics, however, including turbidity, BOD, nitrate, phosphate and COD fell short of the recommended guidelines. All the 48 samples analysed for bacteriological qualities tested positive for the presence of the bacterial groups with significant (P≤0.05) seasonal variation in their densities. Faecal coliforms were detected in counts ranging from 1 CFU/100ml to 2.7 × 104 CFU/100ml. Presumptive E. coli counts ranged generally between 1 CFU/100ml – 1.4 × 105 CFU/100ml while counts of presumptive Vibrio species ranged between 4 CFU/100ml – 1.4 × 104 CFU/100ml. Molecular identification of the presumptive isolates by polymerase chain reactions PCR gave positive reaction rates of 76.2 percent (381/500) and 69.8 percent (279/400) for E.coli and Vibrio species respectively. The antibiotic susceptibility profiling of 205 PCR-confirmed Vibiro isolates against 18 commomly used antibiotics showed resistance frequencies ranging from 0.5 percent (imipenem) to 96.1 percent (penicillin G) at recommended breakpoint concentrations. Eighty-one percent (166/205) of the Vibrio isolates showed multidrug resistance (resistance to 3 or more antibiotics) with the most common multiple antibiotic resistance phenotype (MARP) being AP-T-TM-SMX-PG-NI-PB, occurring in 8 isolates