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Prevalence and genomic characterization of zoonotic Salmonella and selected enteric pathogens in clinical and food samples in Thi-Qar Province, Iraq

Harb, Ali Abdulhadi (2020) Prevalence and genomic characterization of zoonotic Salmonella and selected enteric pathogens in clinical and food samples in Thi-Qar Province, Iraq. PhD thesis, Murdoch University.

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Diarrhoeal diseases remain a major threat to young children in developing countries. Iraq is one such developing country where diarrhoeal diseases have a significant impact on child health, however, due to lack of systematic disease surveillance in Iraq, relatively little is known about the role and public health significance of the enteric pathogens involved in gastrointestinal infections.

On a global scale, non-typhoidal Salmonella is the most important foodborne pathogen due to its widespread dissemination and resistance to antimicrobials used in veterinary and human sectors. The contribution of Salmonella to foodborne diseases in Iraq is, however, unknown. To address this, the overarching aim of this thesis was to determine the prevalence, genomic characterization, zoonotic potential and risk factors of non-typhoidal Salmonella associate with diarrhoeal disease in Iraqi children. A secondary aim was to provide contemporary information on select viral, bacterial and parasitic agents associated with enteric disease using molecular tools.

A cross-sectional study was conducted in Thi-Qar Governorate, south-eastern Iraq, to estimate the prevalence, risk factors and antimicrobial resistance of Salmonella infection associated with diarrhoea. Fresh faecal samples were collected from children under five years of age suffering from diarrhoea who attended children's hospitals. Among 320 stool specimens cultured, 33 (10.3%, 95% CI 8.4–12.4) were positive for non-typhoidal Salmonella spp. Culture positivity for Salmonella was higher in children below two years of age. The source of drinking water (pipe water) (OR 4.7; 95% CI 1.6–13.9), a lower education (illiterate and primary) level of the mother (OR 3.9; 95% CI 1.0–6.4) and exposure to domestic animals (OR 10.5; 95% CI 3.8–28.4) were found to be a significant risk factors associated with Salmonella infection in children in a multivariable logistic regression model. Protective factors that decreased the risk of Salmonella infection included boiling water, exclusive breastfeeding and mother’s hand washing before feeding the child and after cleaning child defecation. Antimicrobial susceptibility tests of the Salmonella isolates demonstrated high levels of resistance to tetracycline (78.8%), azithromycin (66.7%), ciprofloxacin (57.6%), trimethoprim/sulfamethoxazole (51.5%), streptomycin (48.4%), and nalidixic acid (45.5%).

Prompt and appropriate healthcare-seeking is one of the factors associated with reducing diarrhoea duration, which then reduces the possibility of dehydration and its complications, and also avoidance of unnecessary use of antimicrobials. A total of 500 mother-child pairs were interviewed using a structured questionnaire to determine the socio-demographic factors associated with maternal health-care seeking practices in order to improve the maternal management of the diarrhoeal disease. Multivariable logistic regression model of predictor factors indicated that mothers aged >25 years were found to have a much lower odds (OR 0.4, 95% CI 0.2–0.6; P = <0.001) of reported childhood diarrhoea compared to those younger. Almost half of the interviewed mothers (52.4% (262/500)) preferred self-ordered medicine from a pharmacy as an alternative management option for diarrhoea. Relative to mother’s health-care seeking practices with a university education, those with high school education had more possibility of seeking medical center (relative risk ratio (rrr) = 2.4) and pharmacy (rrr = 3.7) as against no treatment.

Faecal samples were further screened using PCR to investigate the prevalence of common enteric pathogens known to be associated with diarrhoea in children, and determine potential risk factors. The most frequently identified pathogen in the faecal samples was adenovirus (34.2 %) followed by Salmonella spp. (14.8%), Entamoeba spp. (13.5%), and Campylobacter spp. (10.9%). Of interest, co-infection with a bacterial, viral, and a parasitic etiologic agent all together was detected in nine cases (9/155 (5.8%)). A logistic regression analysis indicated that children who exclusively breastfed had lower odds of adenovirus DNA detection (P = 0.051; OR 0.46, 95% CI 0.21–0.99) compared to children exclusively bottle-fed. Campylobacter infection in children from households supplied by pipe water was 5.12 (95% CI 1.12–23.27) times higher (P = 0.034) compared with those supplied with treated water. Entamoeba infection was three times lower [P = 0.030, OR 0.34 (95% CI 0.14–0.90)] in children belonging to caregivers who reported always washing hands after cleaning children following defecation, versus those belonging to caregivers who did not wash hands. In addition, for the first time in Iraq, whole genome sequence (WGS) analysis data for 23 Salmonella isolates in the present study indicated that S. Typhimurium ST49 was the most frequent sequence type, and floR, blaCARB-2 and mphA antimicrobial resistance genes were identified for the first time in Salmonella isolated from children.

Chicken meats may play the potential role as sources/carriers of human Salmonella pathogenic strains in the zoonotic transmission and epidemiology of salmonellosis. To evaluate the role of chicken in causing human salmonellosis, 400 frozen chicken carcasses imported from Iran, Turkey, Brazil and India were collected from various retail stores and markets around Thi-Qar to determine the occurrence, serotypes distribution, phenotypic and genotypic characteristics of antibiotic resistance, and multilocus sequence types (ST) of Salmonella. Overall, the frequency of Salmonella contamination was 11.5% (95% CI 8.5%–15.0%) using culture techniques with PCR confirmation. Of the 46 Salmonella species, S. Typhimurium [23.9% (11/46)] and S. Enteritidis [21.7% (10/46)] were the most predominant serotypes. All of the Salmonella isolates tested exhibited resistance to one or more antimicrobial agents used, with common resistance being to tetracycline (84.4%), nalidixic acid (80.4%), streptomycin (69.6%) and trimethoprim/sulfamethoxazole (65.2%). This pilot study provided the first reporting on the genomic characterization of Salmonella isolates from imported chicken carcasses in Iraq. Eight sequence types (STs) were assigned by multilocus sequence typing (MLST) for 40 Salmonella isolates. The most frequent STs among S. Enteritidis was ST-11 and among S. Typhimurium was ST-19. WGS analysis also revealed that Salmonella isolates harboured resistance gene profiles which correlated with their resistance phenotypes. For β-lactamase genes, 18 isolates carried four β-lactamase resistance genes, with blaCARB-2 was the most commonly (14/18) seen while fifteen isolates carried the florfenicol resistance gene (floR) among the 46 isolates. Among nontyphoidal Salmonella isolated from chicken carcasses, 84.8% (39/46) were classified as with multiple drug resistance (MDR). These results indicate that chicken meat may acts as the reservoir for multidrug-resistant Salmonella infection in foodborne exposure to human.

In summary, this study provides baseline carriage of pathogens responsible for causing diarrhoea in children under the age of five in Iraq. In addition, the present study for the first time provides insights into the genomic characteristics of Salmonella isolates collected from children and chicken meat. It also details the distribution of multidrug-resistant (MDR) profiles and resistance genes among Salmonella isolates to the first line antimicrobials regularly used in veterinary and human sectors, with potential for transfer to humans. Furthermore, the identification of common enteric pathogens causing diarrhoea in children will inform future epidemiological conservation studies. The household survey also identified areas to improve the management of diarrhoea in children under the age of five. The outcomes of this thesis may be used to inform public health and zoonotic disease programs for formulating appropriate recommendations for the management of diarrhoea and food safety.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
United Nations SDGs: Goal 2: Zero Hunger
Goal 3: Good Health and Well-Being
Supervisor(s): Abraham, Sam, Habib, Ihab and O'Dea, Mark
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