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Title: Neonatal enteral feeding tube as loci for Enterobacteriaceae colonisation and risk to neonatal health
Author: Abudalla, Halema
ISNI:       0000 0004 5353 1008
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2014
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The incidence of neonatal infections caused by Enterobacteriaceae has been increasing in recent years, and they are now recognised as the predominant causative agents in neonatal intensive care unit (NICU) outbreaks. Klebsiella spp. and Serratia spp. are the most common causative pathogens, and E. coli is one of the leading causes of neonatal meningitis and sepsis. The infant intestinal flora is influenced by the feeding regime. This study focuses on assessing the risk to neonates from the ingestion of the Enterobacteriaceae such as; Enterobacter hormaechei, Enterobacter ludwigii, Enterobacter aerogenes, Enterobacter cloacae and Klebsiella oxytoca. The strains under study were isolated from two sources; human mastic breast milk (MBM) and neonatal nasogastric enteral feeding tubes (EFT). The overall aim was to evaluate the risk to neonates posed by the ingestion of these organisms either from contaminated breast milk or from infant formula. Due to the lack of adequate source information, it was necessary to first confirm the identity of the strains under investigation. This was achieved using standard biochemical profiles (phenotyping) and where necessary 16S rDNA sequence analysis. Secondly, it was necessary to determine whether all strains were unique or if any were multiple isolations of the same strain. This was achieved using Pulsed-Field Gel Electrophoresis (PFGE). To determine the potential exposure of neonates to these organisms, a range of physiological and virulence related assays were undertaken; heat tolerance to 55°C, biofilm formation, capsule formation and acidic pH survival (pH 3.5). The potential virulence of the strains was assessed using attachment-invasion assays of human Caco-2 intestinal cells, human brain microvascular endothelial cells (HBMEC) and rat brain capillary endothelial cell line (rBCEC4); and also persistence of bacteria in macrophages by using U937 cells. Patterns of adherence of Enterobacteriaceae to Caco-2 cells was investigated. The presence of the virulence factors of strains was determined by identifying haemolytic activity, serum resistance, siderophore production and antimicrobial susceptibility. The iron uptake genes were also investigated. The results by PFGE showed that neonatal enteral feeding tubes and mastic human breast milk were contaminated by twenty-one and three pulsotypes of Enterobacteriaceae, respectively. Furthermore, the same pulsotypes were spread among enteral feeding tubes of infants in the same NICUs; indicating the same origins, such as: environment, milk or carer. Similarly, the MBM strains were isolated from the same mother. The identification of strains by using 16S rDNA sequence analysis (genotyping) was more accurate than phenotyping (API technique) and the clustering of strains by PFGE is a suitable technique for strains relatedness. The physiological features of the strains in the current study were investigated. The ability of strains to survive at 55ºC was studied and most of the strains were able to survive at 55ºC for >30 minutes. Biofilm formation was investigated as this may be a factor of organism persistence in the neonatal intensive care unit (via milk, environment or workers) and attachment to enteral feeding tubes. All strains formed biofilms and this was, in general, enhanced at 37°C compared with room temperature (20ºC) in all types of formula. The highest levels of biofilm were in casein-based infant formula. Most strains produced capsular material at 37ºC on all types of formula. However, capsular material was produced by all strains in soya infant formula. All strains were able to survive at pH 3.5 for up to 2 hours. All strains were able to attach to Caco-2, HBMEC and rBCEC4 cells lines, while there was variation between strains ability to invade mammalian cells. In particular, most of Ent. hormaechei strains were able to invade the three types of cells lines and one Ent. ludwigii strain 1439 was only able to invade the rBCEC4 cell line. Ent. ludwigii strain 1439 was isolated from a case of neonatal meningitis. Three out of eight strains of Ent. hormaechei and two strains of Ent. cloacae strains survived within macrophages. Haemolysin production, serum resistance and siderophore production were also studied and all strains were positive. Genes encoding for iron uptake irp1, irp2 and fyuA were detected whereas irp1, irp2 genes were absent in all strains while fyuA was present in 4/6 of Ent. ludwigii strains, 1/8 of Ent. hormaechei, 2 of Ent. cloacae, 1 of Ent. aerogenes and 3/6 of K. oxytoca. Three out of eight strains of Ent. hormaechei showed resistance to even the 3rd generation cephalosporins, ceftazidime and cefotaxime and were ESBL-positive.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available