Browsing by Author "Paul Orndorff, Committee Member"

Now showing 1 - 5 of 5

Deducing the Biological Relevance and Transfer of Antimicrobial Resistance Factors in Salmonella enterica serovar Typhimurium.
(2003-08-11) Leming, Christopher Lloyd; Paul Orndorff, Committee Member; Stephen Libby, Committee Member; Craig Altier, Committee Chair
Antimicrobial resistance in Salmonella can be encoded on plasmids or as stable chromosomal elements. To study the nature of resistance transfer by Salmonella enterica serovar Typhimurium strains of animal origin, we examined isolates obtained from pigs in commercial swine operations. We found that one common phage type, DT193, was most often resistant to five antimicrobials (ampicillin, kanamycin, streptomycin, sulfonamides, and tetracycline). Isolates of this phage type typically encoded all of their resistance genes on conjugative plasmids that could be efficiently transferred to E. coli. One of the DT193 isolates (strain UT20), however, carried its resistance factors integrated into its chromosome. Despite the chromosomal location of these resistance factors, they could still be readily transferred by conjugation and resided in transconjugants on an autonomously replicating plasmid. We also found a spontaneous mutant of UT20 that remained resistant, but could not transfer resistance factors by conjugation, and found mutants having different resistance patterns, each having lost regions of the chromosomally encoded resistance element. Furthermore, the method of plasmid transfer was complex: approximately one-half of E. coli transconjugants encoded all five resistances on a ~140 kb plasmid. Approximately half encoded resistance to kanamycin, tetracycline, streptomycin, and sulfonamides on a plasmid of ~126 kb, while less than 2% had a ~109 kb plasmid encoding resistance to ampicillin alone. Restriction mapping of these three plasmids showed them to be closely related, and all encoded their resistances with identical alleles. We propose that recombinational events in the Salmonella host strain produce these three distinct derivative plasmids. Although the use of antimicrobials in food–producing animals has induced resistance in pathogenic bacteria, it remains unclear whether the cessation of antimicrobial use would eliminate these resistant strains. An important aspect to this question is the growth rate of resistant strains in comparison to similar susceptible strains. To test the fitness for growth of resistant Salmonella, we compared the growth rate in mixed cultures of multi–resistant Salmonella isolates of the DT193 phage type to isogenic fully susceptible derivatives. Competing strains were grown by serial transfer in antimicrobial-free media for 22 days, with each strain in the population quantified every three days. Wild type strains varied greatly in their relative fitness. UT20 displays a great fitness cost when it is in competition against its isogenic, fully susceptible derivative. UT8, whose resistance factors are identical to those of UT20 but are carried on a plasmid, displays an initial fitness cost that is much less severe and appears to be surmountable. This difference in fitness is not due to differences in the genetic backgrounds of the two strains since cured UT20 carrying the plasmid of UT8 also showed only a mild fitness defect. Conjugation within competitions was measured and found not to be an influencing factor on the persistence of resistance factors in a population. It is our conclusion that the degree to which the fitness burden manifests itself is dependent on the location of the resistant element. Naturally occurring multiple drug resistant (MDR) strains of Salmonella enterica are at a large metabolic disadvantage compared to susceptible isogenic strains if the resistance element is integrated into the chromosome. It appears that if the resistance element is in plasmid form, then the fitness burden is significantly less, as well as being surmountable through means as yet undetermined.
Enzyme Properties and mRNA Expression of an NAD+ Scavenging System: NatV and NadV of Vibrio parahaemolyticus Phage KVP40
(2008-08-26) Lee, Jae Yun; Eric Miller, Committee Chair; Paul Orndorff, Committee Member; Jon Olson, Committee Member; Amy Grunden, Committee Member
Genetic and Environmental Mediators of Salmonella Infection
(2007-11-08) Huang, Yanyan; Paul Orndorff, Committee Member; Craig Altier, Committee Co-Chair; Wondwossen A. Gebreyes, Committee Member; Jay F. Levine, Committee Co-Chair
The purpose of the research is to identify genetic determinants and environmental signals of infection in Salmonella enterica serovar Typhimurium. We established pigs as an animal model to identify Salmonella genes specifically expressed in vivo by using a recombinase-based in vivo expression system. A total of 55 clones from a genomic library of ˜ 10,000 random Salmonella DNA fragments were isolated from the tonsils and small intestinal tracts of pigs. Characterization of in vivo induced genes by sequencing showed that genes involved in adhesion, colonization, virulence, stress response, and a two-component regulator were specifically induced after infection of pigs. High temperature and osmolarity induced a number of these in vivo expressed genes. We identified formate as an environmental signal that induces invasion gene expression in Salmonella. The effect of formate required a pH below neutrality, and we found that the distal ileum of mice had the appropriate formate concentration and pH to elicit invasion of Salmonella. We further found that formate plays a role in inducing invasion gene expression by changing carbon flux. To identify the formate regulon, Salmonella DNA microarrays were employed to compare gene expression of wild type Salmonella grown with or without formate. A part of formate hydrogenlyase complex, encoded by the hyc operon, was induced by the additional formate. A large class of genes involved in the respiratory electron transfer system was also affected by formate. Nitrate reductase and succinate dehydrogenase were repressed, while fumarate reductase was induced by formate. Thus, in the presence of formate, fumarate may be a more preferred electron acceptor than nitrate. A number of genes involved in vitamin B12 synthesis, aromatic acid synthesis, flagella synthesis, lipopolysaccharide synthesis, and two-component regulators were also affected by formate. Surprisingly, genes affected by formate overlapped with some in vivo induced genes, suggesting that formate present in the distal ileum could be a signal for Salmonella infection of animal hosts.
Molecular Characterization of Rickettsial Diseases in Dogs
(2006-07-26) Kidd, Linda Benjamin; Robert Kelly, Committee Member; Paul Orndorff, Committee Member; Ed Breitschwerdt, Committee Chair; Wayne Tompkins, Committee Member; Scott Laster, Committee Member
Spotted Fever Group Rickettsia are important causes of morbidity and mortality worldwide. These arthropod borne, obligately intracellular organisms are notoriously difficult to detect in blood samples from infected patients. Standard diagnostic techniques do not differentiate among SFG members, thus the species of infecting Rickettsia is presumed based on geographic location. With the advent of molecular biology, newly discovered and previously known pathogenic and "non-pathogenic" SFG Rickettsia have been associated with disease in people in expanding regions of the world. R. rickettsii, the cause of Rocky Mountain Spotted Fever, is arguably the most well known and well characterized SFG Rickettsia The manifestations of this disease are similar in dogs and people, and because infection can precede or occur simultaneously with infection in their human companions, dogs are considered sentinels for the disease. Despite this, the species of Rickettsia infecting dogs with RMSF had never been characterized using molecular techniques. Using PCR, we amplified and sequenced portions of three genes from SFG Rickettsia isolated from dogs and people with RMSF in an endemic region. Gene sequence based criteria were applied and the isolates were identified as R. rickettsii. This study provides support that naturally occurring RMSF in dogs is comparable to the disease in people. Rickettsia rickettsii has been declared a Select Agent by the CDC and a Category C priority pathogen by the NIAID due to concerns that it is amenable for use in a bioterrorist attack. As a result of this study, 15 consensus sequences of genes amplified from R. rickettsii naturally infecting dogs and people in North Carolina have been deposited in GENBANK (Accesion numbersDQ15680-DQ15694). Knowledge of sequences of naturally occurring isolates may help identify aberrant strains intentionally released as a result of an act of bioterrorism. Amplifying DNA from cultures of Rickettsia does not require a particularly sensitive PCR. Due to the nature of these organisms, sensitive assays are required in the clinical setting. No diagnostic tool that is sensitive, and can differentiate among species of infecting Rickettsia has been validated for use in infected dogs. We have created a PCR that amplifies a portion of the ompA gene from infected dog blood with a limit of detection of 1.5-30 copies of SFG Rickettsia. Sequencing of the product differentiates a number of different species. Using this tool we documented the presence of R. conorii ssp conorii DNA in the blood of three male Sicilian Yorkshire Terriers. These dogs had clinical illness compatible with acute rickettsiosis. This provides unique evidence that R. conorii may infect dogs and cause disease in this host. Future studies should investigate the role of R. conorii and other SFG Rickettsia as disease causing agents in dogs. Identifying SFG Rickettsia in naturally infected dogs may have implications for their human companions due to their potential role as sentinels for this group of illnesses.
Natural Transformation-mediated Transfer of Erythromycin Resistance in Campylobacter coli and Campylobacter jejuni
(2005-11-09) Kim, Joo-Sung; Sophia Kathariou, Committee Chair; Fred Breidt, Committee Member; Donna K. Carver, Committee Member; Paul Orndorff, Committee Member
Campylobacter is the most common bacterial agent causing human gastroenteritis and is mainly transmitted through foods. Campylobacter is a zoonotic agent, and commonly colonizes poultry and other meat animals. Whereas erythromycin resistance in Campylobacter jejuni rarely occurs, erythromycin resistance in Campylobacter coli from meat animals is frequently encountered, and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in C. coli has been associated with a point mutation (A2075G) in the 23S rRNA gene, acting synergistically with the CmeABC efflux pump. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli used as donor. In addition, we studied the effects of environmental factors and species (C. coli vs. C. jejuni), and fitness costs of erythromycin resistance in transformants. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli from turkeys than in swine-derived strains (P<0.01) with frequency of transformation 10⁻⁻⁴ to 10⁻⁶ in turkey-derived strains, but 10⁻⁷ or less in C. coli from swine. Transformants harbored the point mutation, A2075G in the 23S rRNA gene. Erythromycin resistance was stable in transformants following serial transfers, and most transformants had high MIC values (>256μg/ml), as did the C. coli donor strains. In contrast to results obtained with transformation, spontaneous mutants had relatively low erythromycin MIC (32-64μg/ml) and lacked the A2075G mutation. Temperature profoundly affected frequency of transformation to erythromycin resistance in C. coli and transformation frequency at 42°C was significantly higher than at 25°C, 32°C and even 37°C. However, transformation to nalidixic acid resistance was not significantly affected by temperature. No significant difference in transformation frequency was detected between microaerobic (5-10% CO₂) and aerobic conditions. Starvation conditions did not affect transformation frequency to nalidixic acid resistance. Increasing incubation time from 3-4h to 15-17h significantly increased transformation frequency to erythromycin resistance (P<0.05). Transformation of C. jejuni using genomic DNA from erythromycin resistant C. coli revealed that transformation frequency of C. jejuni to erythromycin resistance was lower than C. coli, suggesting that erythromycin resistance in C. coli may not be disseminated via transformation in C. jejuni as frequently as in C. coli. Transformants derived from C. jejuni, however, had high erythromycin MIC values (>256μg/ml) and harbored the A2075G transition, similarly to C. coli transformants. When grown separately at 42°C, an erythromycin-resistant transformant derived from C. coli strain 961 had a similar growth rate as its erythromycin-sensitive parental strain, whereas an erythromycin-resistant transformant derived from C. jejuni strain SC49 had a significantly longer generation time compared to its parental strain. In competitive growth studies, however, the C. coli transformant was at competitive disadvantage in relation to its parental strain in stationary phase, whereas the C. jejuni transformant was at a slight fitness advantage after 14days. Furthermore, in the mixed culture the generation time of the C. jejuni transformant was not significantly different from that of the parental strain. In conclusion, natural transformation has the potential to contribute to dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals and temperature can greatly affect transformation to erythromycin resistance, but not to nalidixic acid resistance. These findings suggest that ecological attributes may play an important role and exert differential impact on the potential of the organism to acquire antimicrobial resistance determinants via natural transformation. However, further study is necessary to characterize the fitness of erythromycin resistant transformants in Campylobacter and identify possible mechanisms underlying the relatively low frequency of erythromycin resistance in C. jejuni.