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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.

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

In recent years, it has been reported that numbers of Escherichia coli increase significantly following centrifugation of sludge during the treatment process. E. coli is used as an indicator of the microbiological quality of sludge-derived products destined for agricultural recycling and of the efficacy of the sludge treatment processes. The re-growth phenomenon is of concern because of the potential for additional treatment requirement / higher disposal costs and loss of consumer confidence associated with a compliance failure. It is hypothesised that a competitive exclusion treatment could be the solution wherein the digestate be exposed to a ‘probiotic’ or defined mixture of micro-organisms, to effectively out compete or eliminate any resident E. coli remaining following treatment. The competitive exclusion principle as a treatment method has already seen application in various industrial sectors, the most well-known being the poultry industry. In experiments it was determined that an antimicrobial producing organism would be most likely to succeed. From the candidates screened, Lactobacillus reuteri proved the most promising. L. reuteri is a known producer of reuterin in the presence of glycerol and organic acids as a part of its normal metabolic activity. In sludge derived nutrient broth in the presence of glycerol and low pH, L. reuteri addition resulted in a reduction of E. coli to undetectable levels. In sludge cake under the same conditions, L. reuteri was less successful. However the addition of glycerol and L. reuteri to sludge cake restricted E. coli growth to a 2 log increase from the initial concentration of E. coli recorded following pasteurisation (an average of around 1x102 cfu/gDs), in comparison in the positive control a 4 log increase was recorded. From this result the sludge cake could be defined as conventionally treated. It can be concluded that competitive exclusion and L. reuteri show promise as a treatment for reducing E. coli re-growth in sludge cake

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