Studies of the nature of Burkholderia cepacia in cystic fibrosis

Burkholderia cepacia is an opportunistic pathogen that colonises of the lungs of cystic fibrosis (CF) patients, with a frequently fatal outcome. Antibiotic resistance is common and highly transmissible epidemic strains have been described in the UK. 37 B. cepacia isolates from clinical and botanical sources were characterised via metabolic capabilities, antibiotic sensitivity, fatty acid methyl ester (FAME) profiles restriction digest analysis of chromosomal DNA by pulsed-gel electrophoresis (PFGE) (with the use of two separate restriction enzymes) and outer membrane protein (OMP) profiles. This revealed isolates of the UK CF epidemic strain to form a distinct group with a specific OMP profile. Cluster analysis of PFGE and FAME profiles revealed the species Burkholderia gladioli and Burkholderia vietnamiensis to be more closely related to each other and to laboratory strains of B. cepacia than to the CF epidemic strain considered a member of the latter species. The epidemic strain of B. cepacia may therefore be worthy of species definition in its own right. All the strains studied showed a high level of resistance to antibiotics, including the carbapenems. Considering this, carbapenemase production by isolates of B. cepacia was investigated. A metallo-β-lactamase from a clinical strain of B. cepacia was isolated and partially purified of using Cibacron blue F3GA-coupled agarose. The resulting preparation showed a single band of β-lactamase activity (pI 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidime, benzylpenicillin, ampicillin and carbenicillin were hydrolysed at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylene diamine tetra acetic acid and o-phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst >90% inhibition was attainable with 0.1mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed an enzyme of pI 8.45 to be present and inducible by imipenem in each case. This enzyme was assigned PCM-I (Pseudomonas cepacia metalloenzyme I).

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays