<p>Bacteria are responsible for up to 70% of all ocular infections including conjunctivitis, keratitis and endophthalmitis. If left untreated, a reduction of visual acuity, and in severe cases, sight loss, is possible. Treatment usually consists of a topically applied antibacterial preparation for patients with superficial infections. With intraocular infections, topical administration is augmented with systemic treatment or local instillation. While several types of drugs are available for ocular therapy, the fluoroquinolone class of antimicrobials is especially effective. This is due in part to their broad-spectrum of activity and low toxicity. However, as with any globally prescribed antimicrobial agent, bacterial resistance is an issue. Over the past 10 years there has been a decline in the effectiveness of older fluoroquinolones (ciprofloxacin and ofloxacin) in treating Gram-positive and, to a lesser extent, certain Gram-negative infections. In response to the declining activity of ciprofloxacin and ofloxacin, newer fluoroquinolones have been developed such as levofloxacin (L-isomer of ofloxacin), and more recently, gatifloxacin and moxifloxacin. In order to ensure the most potent drugs are being used to treat the most serious types of infection, studies need to be done to assess the activity of the current antimicrobial arsenal against pertinent infecting organisms. Three different types of experiments can be done to achieve this. In vitro potency can be tested two ways. The first is minimum inhibitory concentration (MIC). This test defines the concentration of antimicrobial drug that prevents growth of bacteria when tested against an inoculum of approximately 105 colony forming units (CFU)/ml. The second is the mutant prevention concentration (MPC), which is the amount of drug needed to inhibit a first step resistant mutant. This is a relatively new approach to measuring fluoroquinolone potency; like MIC it is not a measure of kill. A separate set of experiments are needed to assess in vitro killing. Kill curves measure the ability of an antimicrobial agent to reduce/kill a bacterial population over a period of 24 hours.</p><p>Because bacterial loads can vary greatly in in vivo infections, kill curves were conducted on a series of four inoculum sizes ranging from 106 to 109 cfu/ml. Some of the most common ocular pathogens are <i>Streptococcus pneumoniae</i>, <i>Staphylococcus aureus</i>, <i>Haemophilus influenzae</i> and <i>Pseudomonas aeruginosa</i>. <i>Mycobacterium fortuitum</i> and <i>Mycobacterium chelonae</i>, while much less commonly associated with ocular disease, are capable of causing vision-threatening infections. As a result, the above six organisms were used to test the in vitro potency of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and gatifloxacin.</p><p>Both MIC and MPC testing found both gatifloxacin and moxifloxacin to be 4-8-fold more potent <i>in vitro</i> against the Gram-positive organisms than the older fluoroquinolones with an average potency rank order of moxifloxacin = gatifloxacin > levofloxacin > ofloxacin = ciprofloxacin. The Gram-negative results, however, revealed that the older fluoroquinolones are still the most potent of the fluoroquinolones tested with an average potency rank order of ciprofloxacin > ofloxacin = levofloxacin > gatifloxacin = moxifloxacin. Kill curve results showed a significant difference in the rate of killing between the MIC and MPC drug concentrations. At the MIC drug concentration there was generally only a noticeable reduction in viable cells following 24 hours of drug exposure and in many cases this was followed by a period of bacterial re-growth. At the MPC drug concentration, a significant bacterial count reduction was often observed as early as 4 to 6 hours for both S. pneumoniae and H. influenzae. Surprisingly, there was little difference between the five fluoroquinolones in their rates of and amount of bacterial reduction.</p><p>Because of high in vitro resistance rates in drugs like penicillin, the fluoroquinolones are an important broad-spectrum alternative. Consequently, it is imperative that measures are taken to maintain the efficacy of this class. One approach is to ensure that the most potent drug is being used to eradicate possible resistant sub-populations present in in vivo infections. The data from these experiments suggest that the new fluoroquinolones gatifloxacin and moxifloxacin are much more potent (<i>in vitro</i>) than older fluoroquinolones against Gram-positive bacteria. With Gram-negative pathogens, however, ciprofloxacin remains the most potent agent <i>in vitro</i>.</p>

<p>Bacteria are responsible for up to 70% of all ocular infections including conjunctivitis, keratitis and endophthalmitis. If left untreated, a reduction of visual acuity, and in severe cases, sight loss, is possible. Treatment usually consists of a topically applied antibacterial preparation for patients with superficial infections. With intraocular infections, topical administration is augmented with systemic treatment or local instillation. While several types of drugs are available for ocular therapy, the fluoroquinolone class of antimicrobials is especially effective. This is due in part to their broad-spectrum of activity and low toxicity. However, as with any globally prescribed antimicrobial agent, bacterial resistance is an issue. Over the past 10 years there has been a decline in the effectiveness of older fluoroquinolones (ciprofloxacin and ofloxacin) in treating Gram-positive and, to a lesser extent, certain Gram-negative infections. In response to the declining activity of ciprofloxacin and ofloxacin, newer fluoroquinolones have been developed such as levofloxacin (L-isomer of ofloxacin), and more recently, gatifloxacin and moxifloxacin. In order to ensure the most potent drugs are being used to treat the most serious types of infection, studies need to be done to assess the activity of the current antimicrobial arsenal against pertinent infecting organisms. Three different types of experiments can be done to achieve this. In vitro potency can be tested two ways. The first is minimum inhibitory concentration (MIC). This test defines the concentration of antimicrobial drug that prevents growth of bacteria when tested against an inoculum of approximately 105 colony forming units (CFU)/ml. The second is the mutant prevention concentration (MPC), which is the amount of drug needed to inhibit a first step resistant mutant. This is a relatively new approach to measuring fluoroquinolone potency; like MIC it is not a measure of kill. A separate set of experiments are needed to assess in vitro killing. Kill curves measure the ability of an antimicrobial agent to reduce/kill a bacterial population over a period of 24 hours.</p><p>Because bacterial loads can vary greatly in in vivo infections, kill curves were conducted on a series of four inoculum sizes ranging from 106 to 109 cfu/ml. Some of the most common ocular pathogens are <i>Streptococcus pneumoniae</i>, <i>Staphylococcus aureus</i>, <i>Haemophilus influenzae</i> and <i>Pseudomonas aeruginosa</i>. <i>Mycobacterium fortuitum</i> and <i>Mycobacterium chelonae</i>, while much less commonly associated with ocular disease, are capable of causing vision-threatening infections. As a result, the above six organisms were used to test the in vitro potency of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and gatifloxacin.</p><p>Both MIC and MPC testing found both gatifloxacin and moxifloxacin to be 4-8-fold more potent <i>in vitro</i> against the Gram-positive organisms than the older fluoroquinolones with an average potency rank order of moxifloxacin = gatifloxacin > levofloxacin > ofloxacin = ciprofloxacin. The Gram-negative results, however, revealed that the older fluoroquinolones are still the most potent of the fluoroquinolones tested with an average potency rank order of ciprofloxacin > ofloxacin = levofloxacin > gatifloxacin = moxifloxacin. Kill curve results showed a significant difference in the rate of killing between the MIC and MPC drug concentrations. At the MIC drug concentration there was generally only a noticeable reduction in viable cells following 24 hours of drug exposure and in many cases this was followed by a period of bacterial re-growth. At the MPC drug concentration, a significant bacterial count reduction was often observed as early as 4 to 6 hours for both S. pneumoniae and H. influenzae. Surprisingly, there was little difference between the five fluoroquinolones in their rates of and amount of bacterial reduction.</p><p>Because of high in vitro resistance rates in drugs like penicillin, the fluoroquinolones are an important broad-spectrum alternative. Consequently, it is imperative that measures are taken to maintain the efficacy of this class. One approach is to ensure that the most potent drug is being used to eradicate possible resistant sub-populations present in in vivo infections. The data from these experiments suggest that the new fluoroquinolones gatifloxacin and moxifloxacin are much more potent (<i>in vitro</i>) than older fluoroquinolones against Gram-positive bacteria. With Gram-negative pathogens, however, ciprofloxacin remains the most potent agent <i>in vitro</i>.</p>

The first aim of this study (Chapter III), the effects of chlorate and nitroethane on foodborne pathogens and rumen fermentation were evaluated. The experimental chlorate product, reduced (P < 0.001) fecal, but not ruminal (P > 0.05) E. coli concentrations by 1000- and 10-fold by 24 and 48 h after chlorate feeding when compared to pre-treatment concentrations (> 5.7 log10 colony forming units/g). Nitroethane treatment decreased (P < 0.01) ruminal (8.46, 7.91 and 4.74 ± 0.78 μmol/mL h-1) and fecal (3.90, 1.36 and 1.38 ± 0.50 μmol/g h-1) methane-producing activity for treatments 0, 80 and 160 mg nitroethane/kg body weight per day, respectively. Whole animal methane emissions, expressed as L/d or as a proportion of gross energy intake (%GEI) were unaffected by nitroethane treatment (P > 0.05). The second aim of this study (Chapter IV) was conducted to examine the effects of nitroethane and monensin on ruminal fermentation and nitro-metabolizing bacterial populations in vitro. The addition of nitroethane decreased methane production (μmol/mL) by at least 90%. The most probable number (MPN) of nitro-metabolizing bacterial populations was increased (P < 0.01) with the addition of nitroethane by at least 3 log10 cells/mL compared with monensin, monensin plus nitroethane or the control group. The final aim of this study (Chapter V) evaluated the effect of two sources of tannins, chestnut (CT) and mimosa (MT) on foodborne pathogens when applied as a hide-intervention and as a feed additive to feedlot cattle. Tannin spray application showed no effect of treatment or application-time (P > 0.05) on E. coli/total coliforms and total aerobes. Chestnut tannin decreased bacterial load of ruminal E. coli and total coliform by at least 0.4 log10 CFU/mL. However, fecal E. coli concentrations were increased with mimosa by 0.3 log10 CFU/g. Also, fecal total coliforms increased with the addition of chestnut or mimosa by at least 0.3 log10 CFU/g. Fecal Campylobacter concentrations (log10 CFU/g) increased with the addition of chestnut and mimosa by at least 0.4 log10 CFU/g.

No description available.

No description available.

No description available.

Studies were undertaken to examine the extent of molecular variation among isolates of the fish pathogen Renibacterium salmoninarum. As many isolates as possible were gathered from diagnostic laboratories within the UK, and checked for viability and contamination. The isolates were derived mainly from infected rainbow trout and Atlantic salmon that were farmed in England, Scotland and Wales, subject to the requirements of statutory legislation. Incomplete histories were available for the sources of the isolates, which spanned a time scale of 36 years, from 1962-1998. Molecular variation between the isolates was examined using two strategies. Firstly, defined regions of the genome were examined for polymorphisms. PCR analysis of previously characterised genes, msa, hly, and rsh, revealed no length polymorphisms among 43 UK isolates of R. salmoninarum. The 23S and 5S rRNA genes were sequenced and sequence analysis of the 16S- 23S (ITS I) and 23S-SS (ITS2) rRNA regions was performed. Sequence analysis confirmed the correct taxonomic placement of R. salmoninarum within the Micrococcus/Arthrobacter subdivision of the Actinomycetes. Some isolates possessed small sequence variations within ITS1 that can provide an indication of their geographical origins. Sequence variation also exists in the ITS2 region but was found only within a single isolate from an outlying region of Canada. Ribotyping was found to be of limited use for discriminating among isolates of R. salmoninarum probably because R. salmoninarum possesses only two copies of the rRNA operon with no length polymorphisms in the ITS regions. Additionally, the discovery and analysis of a genetic locus containing a 51 bp exact tandem repeat unit (designated ETR-A), revealed that some isolates of R. salmoninarum could be distinguished by the possession of one rather than two copies of this repeat unit. Finally, PCR amplification of length polymorph isms in the tRNA gene spacer regions (tDNA-PCR) was developed using consensus tRNA gene primers to enable tRNA genes and spacer regions to be cloned and sequenced. Subsequently, specific tRNA gene primers were designed and enabled the discrimination of 43 UK isolates into I 5 groupings. tDNA-PCR proved to be one of the most powerful typing methods applied to this organism. Secondly, typing methods that analysed the whole genome for the presence of molecular variation were employed. A putative insertion sequence IS994, was used as a probe in a RFLP-based study to discriminate between 52 isolates of R. salmoninarum from different countries. This method showed great potential for distinguishing between isolates and separated the 52 isolates that were examined into 12 groupings. Randomly amplified polymorphic DNA (RAPD) was also used to examine 28 isolates of UK origin. This method was found to be highly discriminatory, with 28 isolates generating 12 different banding patterns, which appeared to reflect geographical source. Pulsed field gel electrophoresis was also used to investigate the genomic diversity of isolates. Due to technical difficulties in obtaining pure, undamaged DNA the preparation of suitable macro restriction profiles was never achieved. However, preliminary findings suggested that the R. salmoninarum chromosome was linear and approximately 4.5-6Mb in size. Finally, repetitive element PCR was evaluated using 3 different approaches (ERIC, REP and BOXA-2) but proved to have a limited capacity for defining molecular variation between different isolates. Ultimately, RAPD, tDNA-PCR and 1S994 RFLP profiling proved to be most useful for the molecular differentiation of R. salmoninarum. A comparison of the groupings that resulted from each of these methods revealed substantial areas of agreement. The use of a multifactorial approach not only resulted in a greater discrimination of isolates but also provided increased confidence in the outcome. It is recommended that for typing purposes such an approach should be adopted. Epizootiological interpretations of groupings were hampered by the general lack of background information attached to each isolate. However, the application of multiple typing methods reveal that, despite the highly conserved nature of this bacterium, UK isolates of R. salmoninarum possess genetic diversity, with geographically related isolates often being grouped together. Overall there was little evidence to suggest the regular introduction of genetically distinct R. salmoninarum into the UK and the results suggest that some isolates may be relatively localised despite the international trade in fish stocks.

No description available.

Thesis (Ph.D.)--Boston University / The role of specific streptococcal products in the production of infection by these organisms has been the subject of many studies by previous investigators. To a great extent these workers have attempted to correlate the formation of these substances by certain streptococcal strains with their virulence and disease manifestations in man as well as with the lethal effects produced by these organisms in mice. The purpose of this study was to investigate further the role of individual streptococcal products in the pathogenicity of group A streptococci, mainly by observing the effects of these products on local and systemic infections in the hamster and the mouse, respectively. Specifically, the M protein, streptokinase, streptodornase, streptolysin 0, the erythrogenic toxin, the hyaluronic acid capsule, and hyaluronidase were studied. [TRUNCATED]

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated which are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for the intracellular growth defect, colony morphology on LB (X-p) media, and virulence in mice. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. Preliminary studies have also identified a convergently transcribed gene, tentatively designated mglC, immediately downstream of mglB. mglC null mutants are defective for intracellular growth and show the same phenotype on LB (X-p) agar as GB2. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Western blot analysis revealed the absence of MglA in an mglB null mutant, indicating MglB may influence MglA levels. Analysis of the regulation of mglA expression during growth in broth culture shows a decrease in expression upon entering late log-early stationary phase. mglA is also expressed during culture in macrophages. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida, most notably the absence of a 70 kDa secreted protein. A candidate clone for the gene encoding this 70 kDa protein has been isolated. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may control the expression of genes whose products contribute to survival and growth within macrophages. Roles for the putative MglC and possibly the 70 kDa secreted protein in this activity are also indicated. Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. The gene encoding AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella , was cloned and sequenced. The deduced amino acid sequence of AcpA showed limited similarity to phospholipase C proteins present in Pseudomonas aeruginosa and Mycobacterium tuberculosis. An F. novicida acpA null mutant was found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages as well as remaining virulent for mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida, and that any role it may play in virulence is subtle. / Graduate