The soil-dwelling gram-positive coccobacillus Rhodococcus equi is a well-known veterinary pathogen and emerging human pathogen. Although Rhodococcus infection is primarily associated with pyogranulomatous pneumonia in foals, these bacteria can also infect other animal species including humans. R. equi pathogenicity is mediated by the conjugative virulence plasmid (pVAP) which promotes intracellular proliferation in host macrophages. Currently R. equi is endemic in horse breeding farms worldwide. No R. equi vaccine is available and both treatment and prophylaxis rely on the administration of a prolonged course with a combination of macrolide antibiotics (typically erythromycin) and rifampicin. These antimicrobials were introduced in the therapy against R. equi in the 1980s and multiresistance has now emerged among foal isolates, increasing the risk of zoonotic transmission. In this thesis, the role of conjugal extrachromosomal replicons in the host adaptation of R. equi was explored. A previous epidemiological study indicated that different variants of the pVAP virulence plasmid are associated with different animal hosts. This PhD project provides experimental confirmation of a R. equi plasmid-driven host tropism. In vivo and in vitro competition assays were performed using a set of isogenic strains only different in the virulence plasmid type, in adapted (horse) and non-adapted (mouse) model species. The data obtained in the horse model provides clear evidence of a significant negative selection of the non-equine virulence plasmids both at a cell and at the animal level, while no selection was observed in the non-adapted mouse model. Furthermore, this project characterized the determinant responsible for macrolide resistance in R. equi, a novel erm methylase gene, erm(46). The erm(46) determinant was shown to be transferable between strains by conjugation and herein the underlying mechanism and how erm(46) becomes stabilized in R. equi is described. PacBio SMRT-sequencing based analysis revealed that the erm(46) gene is carried in a self-replicating conjugative plasmid of about 80 kb, that we designated pRErm46. The conjugation machinery of pRErm46 was hypothesized to be responsible for bringing the erm(46) determinant into R. equi. However, some erythromycin resistant isolates lack pRErm46 but erm(46) transfer is still observed. This reflects the observation that erm(46) is present in a mobile element that, upon acquisition with the pRErm46 replicon, transposes at a high frequency and to multiple locations of the host genome. If the erm(46) mobile element transposes to the chromosome, no further transfer of the resistance is observed at a detectable frequency in the absence of pRErm46. On the other hand, if the erm(46) element transposes to the R. equi virulence plasmid, the erm(46) determinant co-opts the pVAPA conjugal transfer machinery and gets transferred at the same high frequency as the virulence plasmid (10-2). This constitutes a unique example of efficient co-transfer, in the same genetic vehicle, of virulence and antimicrobial determinants, two key niche-adaptive traits required for within-host survival of bacterial pathogen.

N5-59, a siderophore deficient mutant strain of Rhodococcus erythropolis strain IGTS8 (IGTS8) was investigated to learn more about how this poorly characterized bacterium acquires iron. N5-59 cells were starved for iron and then lysed to release any intracellular siderophore. No intracellular siderophore was detected indicating that N5-59 is not defective in the export or release of siderophore but is probably unable to synthesize siderophore. In a cross-feeding bioassay, growth of N5-59 (in an iron depleted medium) was greatly enhanced by the addition of exogenous siderophore from IGTS8 and other Rhodococcus species indicating that N5-59 is not defective in siderophore uptake. A DNA hybridization probe was prepared using DNA flanking the site of insertional mutation in strain N5-59. This probe was then used to clone a 6 kilobase pair, PstI restriction fragment from the chromosome IGTS8. This cloned DNA is expected to contain the intact gene(s) that was interrupted in N5-59.

Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus

Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality.

Acrylonitrile (AN) is a major volatile waste generated in the production of acrylamide and often associated with aromatic contaminants (toluene and styrene) in plant effluents. We examined Rhodococcus rhodochrous DAP 96622 to determine if it could be adapted to efficient biodegradation of acrylonitrile (AN) in a bioreactor. A model bioreactor with granular activated carbon (GAC) as a substratum for Rhodococcus with AN as sole carbon or in combination with toluene was established. The kinetics of AN biodegradation by immobilized and planktonic cells were evaluated and compared. Inlet load and empty retention time were varied to test the removal efficiency in fed-batch and single-pass mode reactor. In addition, the three dimensional structure and characteristics of the biofilm were followed using confocal scanning laser microscopy (CSLM) and relative software. Immobilized cells in the bioreactor, at starting concentrations of AN up to 1150 mg l-1 in the presence of Tol, had at least 13 fold higher AN degradation rates than that seen of planktonic cells. A near steady state of AN degradation was maintained at 75-85% for AN and 80%-90% for Tol within the parameter of EBRT=8 min and AN and Tol inlet loads between 50-200 mg l-1 h-1 and 200-500 mg l-1h-1, respectively. However, when the inlet load of AN was increased to more than 200mg l-1 h-1 and 500 mg l-1 h-1 for Tol, a reduction in efficiency of AN degradation was observed. Biofilms with discrete microcolonies interspersed with voids and channels were observed. Precise measurement of biofilm characteristics agreed with the assumption that the biomass and thickness of the biofilm increased along the carbon column depth. With a porous attachment material like GAC, substrate diffusion is most likely not a limiting factor for AN degradation. Rhodococcus rhodochrous DAP 96622 in a non-sterile activated charcoal column showed efficient degradation of AN in the presence of Tol. The Rhodococcus bioreactor may provide a potential practical waste gas and water treatment system.

Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea). This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions. Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst. The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp. Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

Spurred by a host of beneficial uses, the global use of nanoparticles is rapidly growing. Silver nanoparticles (Ag NPs) are used widely in consumer products, medicine, and the semiconductor industry. As nanoparticles become more commonly used, the transport of nanoparticles into the environment might negatively affect microorganisms in natural and engineered systems. The effects of Ag NPs on microorganisms have primarily been studied in planktonic or free-swimming cultures, but little work has been done to look at biofilm susceptibility to Ag NPs. This thesis describes bacterial tolerance, or the ability of an organism to survive exposure to an insult, to Ag NPs. The tolerance of planktonic and biofilm cells of the common wastewater treatment bacterium Zoogloea ramigera and a Rhodococcus strain isolated from drinking water was tested. These bacteria were exposed to different concentrations of Ag NPs, ranging from 0 to 25 mg/L, for a period of 5 hours. Results showed decreased tolerance with increasing Ag NP concentrations for both bacterial species. Z. ramigera biofilm cells are slightly more tolerant to Ag NPs than are planktonic cells. On the other hand, Rhodococcus planktonic and biofilm cells exhibit similar tolerance. However, in both cases, biofilm cells do not exhibit a striking protective effect against Ag NPs as compared to planktonic cells. This study shows that even short-term insults with Ag NPs can affect bacteria in engineered systems. A preliminary study of the shedding of free silver ions as a possible mechanism of Ag NP toxicity demonstrated that free silver ions were toxic to Escherichia coli in a 0.14M chloride environment. The data suggest that free silver ions can be pulled into solution from Ag NPs in chloride environments via ligand-promoted dissolution. Further work is needed to examine the antibacterial mechanism of Ag NPs against planktonic and biofilm cells to better understand how the release of nanoparticles into the environment can affect microorganisms in natural and engineered water systems. / text