The development of Lactococcus lactis as an antimicrobial agent

Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications. The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363. L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin. Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted. The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T cells

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Anti-infectives

18.1 Antibiotics 18.1.1 Introduction to bacteria 18.1.2 Introduction to antibiotics 18.1.3 Inhibitors of bacterial cell wall synthesis 18.1.3.1 β-Lactams 18.1.3.2 Glycopeptides 18.1.4 Inhibitors of bacterial protein synthesis 18.1.4.1 Tetracyclines 18.1.4.2 Aminoglycosides 18.1.4.3 Chloramphenicol 18.1.4.4 Macrolides 18.1.4.5 Lincosamides 18.1.4.6 Oxalazidones 18.1.5 Inhibitors of DNA synthesis 18.2. Anti-tuberculotic drugs 18.2.1 Introduction 18.2.2 Isoniazid 18.2.3 Ethambutol 18.2.4 Rifamycin 18.2.5 Pyrazinamide 18.3. Anti-viral drugs 18.3.1 Introduction to viruses 18.3.2 Drugs used to treat herpesviruses 18.3.3 Drugs used to treat the flu 18.3.4 Drugs used to treat HIV/AIDS 18.4. Antifungal drugs 18.4.1 Introduction to Fungi 18.4.2 Antifungal drugs

Hybrid antibacterial fabrics with extremely high aspect ratio Ag/AgTCNQ nanowires

This study reports the synthesis of extremely high aspect ratios (>3000) organic semiconductor nanowires of Ag–tetracyanoquinodimethane (AgTCNQ) on the surface of a flexible Ag fabric for the first time. These one-dimensional (1D) hybrid Ag/AgTCNQ nanostructures are attained by a facile, solution-based spontaneous reaction involving immersion of Ag fabrics in an acetonitrile solution of TCNQ. Further, it is discovered that these AgTCNQ nanowires show outstanding antibacterial performance against both Gram negative and Gram positive bacteria, which outperforms that of pristine Ag. The outcomes of this study also reflect upon a fundamentally important aspect that the antimicrobial performance of Ag-based nanomaterials may not necessarily be solely due to the amount of Ag+ ions leached from these nanomaterials, but that the nanomaterial itself may also play a direct role in the antimicrobial action. Notably, the applications of metal-organic semiconducting charge transfer complexes of metal-7,7,8,8-tetracyanoquinodimethane (TCNQ) have been predominantly restricted to electronic applications, except from our recent reports on their (photo)catalytic potential and the current case on antimicrobial prospects. This report on growth of these metal-TCNQ complexes on a fabric not only widens the window of these interesting materials for new biological applications, it also opens the possibilities for developing large-area flexible electronic devices by growing a range of metal-organic semiconducting materials directly on a fabric surface.

Phagocytosis of bacteria by olfactory ensheathing cells and Schwann cells

Opportunistic bacterial infections of the nasal cavity could potentially lead to infection of the brain if the olfactory or trigeminal nerves are colonised. The olfactory nerve may be a more susceptible route because primary olfactory neurons are in direct contact with the external environment. Peripheral glia are known to be able to phagocytose some species of bacteria and may therefore provide a defence mechanism against bacterial infection. As the nasal cavity is frequently exposed to bacterial infections, we hypothesised that the olfactory and trigeminal nerves within the nasal cavity could be subjected to bacterial colonisation and that the olfactory ensheathing cells and Schwann cells may be involved in responding to the bacterial invasion. We have examined the ability of mouse OECs and Schwann cells from the trigeminal nerve and dorsal root ganglia to phagocytose Escherichia coli and Burkholderia thailandensis in vitro. We found that all three sources of glia were equally able to phagocytose E. coli with 75-85% of glia having phagocytosed bacteria within 24h. We also show that human OECs phagocytosed E. coli. In contrast, the mouse OECs and Schwann cells had little capacity to phagocytose B. thailandensis. Thus subtypes of peripheral glia have similar capacities for phagocytosis of bacteria but show selective capacity for the two different species of bacteria that were examined. These results have implications for the understanding of the mechanisms of bacterial infections as well as for the use of glia for neural repair therapies.

Antibacterial anthranilic acid derivatives from Geijera parviflora

Five anthranilic acid derivatives, a mixture I of three new compounds 11′-hexadecenoylanthranilic acid (1), 9′-hexadecenoylanthranilic acid (2), and 7′-hexadecenoylanthranilic acid (3), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid (4) together with a new natural product, hexadecanoylanthranilic acid (5), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1-3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains. © 2013 Elsevier B.V.

Role of capsule and O antigen in the virulence of uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

The radiological diagnosis of pneumonia in children

Despite the importance of paediatric pneumonia as a cause of short and long-term morbidity and mortality worldwide, a reliable gold standard for its diagnosis remains elusive. The utility of clinical, microbiological and radiological diagnostic approaches varies widely within and between populations and is heavily dependent on the expertise and resources available in various settings. Here we review the role of radiology in the diagnosis of paediatric pneumonia. Chest radiographs (CXRs) are the most widely employed test, however, they are not indicated in ambulatory settings, cannot distinguish between viral and bacterial infections and have a limited role in the ongoing management of disease. A standardised definition of alveolar pneumonia on a CXR exists for epidemiological studies targeting bacterial pneumonias but it should not be extrapolated to clinical settings. Radiography, computed tomography and to a lesser extent ultrasonography and magnetic resonance imaging play an important role in complicated pneumonias but there are limitations that preclude their use as routine diagnostic tools. Large population-based studies are needed in different populations to address many of the knowledge gaps in the radiological diagnosis of pneumonia in children, however, the feasibility of such studies is an important barrier.

Cough and reflux esophagitis in children: their co-existence and airway cellularity

Background There are no prospective studies that have examined for chronic cough in children without lung disease but with gastroesophageal reflux (GER). In otherwise healthy children undergoing flexible upper gastrointestinal endoscopy (esophago-gastroscopy), the aims of the study were to (1) define the frequency of cough in relation to symptoms of GER, (2) examine if children with cough and reflux esophagitis (RE) have different airway cellularity and microbiology in bronchoalveolar lavage (BAL) when compared to those without. Methods Data specific for chronic cough (>4-weeks), symptoms of GER and cough severity were collected. Children aged <16-years (n = 150) were defined as 'coughers' (C+) if a history of cough in association with their GER symptoms was elicited before BAL were obtained during elective esophago-gastroscopy. Presence of esophagitis on esophageal biopsies was considered reflux esophagitis positive (E+). Results C+ (n = 69) were just as likely as C- (n = 81) to have esophagitis, odds ratio 0.87 (95%CI 0.46, 1.7). Median neutrophil percentage in BAL was significantly different between groups; highest in C+E- (7, IQR 28) and lowest in C-E+ (5, IQR 6). BAL positive bacterial culture occurred in 20.7% and were more likely present in current coughers (OR 3.37, 95%CI 1.39, 8.08). Airway neutrophilia (median 20%, IQR 34) was significantly higher in those with BAL positive bacterial cultures than those without (5%, 4; p = 0.0001). Conclusion In children without lung disease, the common co-existence of cough with symptoms of GER is independent of the occurrence of esophagitis. Airway neutrophilia when present in these children is more likely to be related to airway bacterial infection and not to esophagitis.

Canine-human transmission of Gastrospirillum hominis

We report electron microscopic evidence of transmission from a pet dog to a 12-year-girl of Gastrospirillum hominis which caused gastric disease in both that was eradicable with treatment. © 1994.

Urinary tract infection of mice to model human disease: Practicalities, implications and limitations

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis

Oxazolidine-2-thiones: A molecular modeling study

Two oxazolidine-2-thiones, thio-analogs of linezolid, were synthesized and their antibacterial properties evaluated. Unlike oxazolidinones, the thio-analogs did not inhibit the growth of Gram positive bacteria. A molecular modeling study has been carried out to aid understanding of this unexpected finding.