Browsing by Author "Craig Altier, Committee Member"

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Erythromycin Susceptibility and Genomic Regions Characterization of Campylobacter coli
(2007-11-01) Chan, Kamfai; Todd Klaenhammer, Committee Member; Fred Breidt, Committee Member; Sophia Kathariou, Committee Chair; Craig Altier, Committee Member
Campylobacter jejuni and Campylobacter coli are major bacterial food-borne pathogens that cause enteric diseases worldwide, resulting in significant public health and economic burden. These two closely related species colonize a wide range of farm animals including turkeys, pigs, chickens, and cattle. Consumption of meat (especially poultry) contaminated with C. jejuni or C. coli has been implicated as the major route of infection. When needed, antibiotics used for treatment are fluoroquinolones or macrolides such as erythromycin. Recent studies have shown that the percentage of C. coli that have resistance to antimicrobials, including those typically used for treatment of human campylobacteriosis, has increased, making it a top priority to investigate the mechanisms for acquisition and dissemination of antimicrobial resistance in these pathogens. Certain Campylobacter strains harbor a transcribed intervening sequence (IVS) in their 23S rRNA genes. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthermore, the distribution of IVS-harboring strains within Campylobacter populations is poorly understood. In this study, strains of Campylobacter coli from turkeys, representing numerous different multilocus sequence typing (MLST)-based sequence types (STs), were characterized in terms of IVS content and erythromycin susceptibility. We identified strains that harbored IVSs in all three 23S rRNA genes, as well as strains lacked IVSs from at least one of the genes. The STs of the latter strains belonged to an unusual cluster of C. coli STs ('cluster II'), earlier found primarily in turkey strains, and characterized by presence of the C. jejuni aspA103 allele. The majority of strains harboring IVSs in all three 23S rRNA genes were resistant to erythromycin. In contrast, cluster II strains, which harbored at least one IVS-free 23rRNA gene, were susceptible to the antibiotic. Cluster II strains could be transformed to erythromycin resistance with genomic DNA from C. coli that harbored IVS and the A2075G transition in the 23S rRNA gene, associated with resistance to erythromycin in Campylobacter. Erythromycin-resistant transformants harbored both the A2075 transition and IVS. The findings suggest that absence of IVS in C. coli from turkeys is characteristic of a unique clonal group of erythromycin-susceptible strains, and that IVS can be acquired by these strains via natural transformation to erythromycin resistance. Analysis of C. coli and C. jejuni strains isolated from broilers, turkeys and swine has shown the association of the lack of IVS and erythromycin susceptibility is unique to C. coli from turkeys. The presence of the C. jejuni aspA103 allele in the chromosome of cluster II C. coli strains is a unique characteristic of this clonal group. To further characterize the genome composition in the aspA region in cluster II strains, we determined the corresponding DNA sequences from two turkey-derived cluster II C. coli strains (6979 and 7474, of ST-1150 and ST-1161, respectively). Genomic organization upstream of the aspA gene was divergent between these two cluster II strains and the reference strain C. coli RM2228, the genome sequence of which has been completed. Genes encoding a putative Crp-family transcriptional regulator (CCO0137) and a conserved hypothetical protein (CCO0138) that were present in C. coli RM2228 and C. coli 6818 were not found in the same genomic region in C. coli 6979 or C. coli 7474. Moreover, single nucleotide polymorphism (SNP) analysis revealed that genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (CJ0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA) and uracil-DNA glycosylase (ung) harbored C. coli-specific SNPs in their 5' portions but C. jejuni-specific SNPs in their 3' portions, suggesting that these may be hybrid genes that were originated from C. jejuni and C. coli. Our data suggest the presence of recombination events in the genomic region between cydA and ung in cluster II strains of C. coli. Such genomic features may contribute to the observed prevalence of cluster II strains among C. coli from turkeys, and to the characteristic susceptibility to erythromycin exhibited by these strains.
Global Effects of the Transcriptional Regulators ArcA and FNR in Anaerobically Grown Salmonella enterica sv. Typhimurium 14028s.
(2008-11-25) Evans, Matthew Richard; Hosni M. Hassan, Committee Chair; Craig Altier, Committee Member; Matthew D. Koci, Committee Member; Eric S. Miller, Committee Member; Jonathan Olson, Committee Member
EVANS, MATTHEW RICHARD. Global Effects of the Transcriptional Regulators ArcA and FNR in Anaerobically Grown Salmonella enterica sv. Typhimurium 14028s. (Under the direction of Dr. Hosni M. Hassan.) The purpose of this research was to assess and compare the genome-wide transcriptional profiles and virulence in mice of the global regulators, FNR (Fumarate Nitrate Reductase) and ArcA (Aerobic Respiratory Control) in anaerobically grown Salmonella enterica serovar Typhimurium 14028s. FNR controls the expression of target genes by sensing and responding to the presence or absence of dioxygen via assembly-disassembly of oxygen-liable iron-sulfur clusters, while ArcA is a two-component (ArcA/ArcB), cytosolic redox response regulator. This work demonstrates that FNR is a positive regulator of motility, flagellar biosynthesis, and pathogenesis. An fnr mutant was non-motile, lacked flagella, attenuated for virulence in mice, and did not survive inside macrophages. In S. Typhimurium, as in Escherichia coli, the FNR modulon encompassed the core metabolic and energy functions as well as motility. Salmonella-specific genes/operons regulated by FNR included those required for ethanolamine utilization, newly identified flagellar genes (mcpAC, cheV), several virulence genes in Salmonella pathogenicity island 1 (SPI-1), and the srfABC operon. ArcA serves as a transcriptional repressor/activator coordinating cellular metabolism and motility. An arcA mutant was non-motile, lacked flagella, and was as virulent as the wild-type strain via intraperitoneal challenge in mice. In S. Typhimurium, as in E. coli, the ArcA modulon encompassed the core metabolic and energy functions as well as motility. Salmonella-specific genes/operons regulated by ArcA included those for propanediol utilization, newly identified flagellar genes (mcpAC, cheV), Gifsy-1 prophage genes, and a few virulence genes located in SPI-3 (mgtBC, slsA, STM3784). Regulation by either ArcA or FNR in S. Typhimurium is similar, but distinct from that in E. coli. Genes/operons involved in the succinyl-CoA pathway, fatty acid degradation, flagellar biosynthesis, motility, chemotaxis, cytochrome oxidase complexes are regulated similarly in the two organisms by ArcA. Genes/operons involved in aerobic metabolism, NOâ€¢ detoxification, flagellar biosynthesis, motility and chemotaxis, and anaerobic carbon utilization are regulated similarly in the two organisms by FNR. Herein, we present the first report on the global role of these two redox regulators in S. Typhimurium. According this study, we hypothesize that FNR plays a more heirarchical role than ArcA in pathogenesis and during the transition between aero- and anaerobiosis in the host. Furthermore, when comparing the motility and virulence results from our work on FNR to those on ArcA, we show that the lack of motility does not necessarily correspond to the lack of virulence in S. Typhimurium.
Molecular Characterization of Listeria monocytogenes Epidemic Clone I (ECI) like isolates from Food and Food Environments
(2007-08-08) Yildirim, Suleyman; Jeffrey Thorne, Committee Member; Craig Altier, Committee Member; Sophia Kathariou, Committee Chair; Lee-Ann Jaykus, Committee Co-Chair; Todd R Klaenhammer, Committee Member
Phenotypic and Genotypic characterization of Campylobacter isolated in swine from Conventional and Antimicrobial free farms
(2005-11-02) Thakur, Siddhartha; Glen Almond, Committee Member; Craig Altier, Committee Member; Wondwossen Gebreyes, Committee Chair; Maria Correa, Committee Member
Campylobacter is the leading cause of bacterial foodborne infections in the world. Campylobacter coli is an important species and ranks second to Campylobacter jejuni in causing clinical infection in humans in the United States. Pigs have been shown to be the major reservoir of this pathogen at the pre-harvest level in conventional swine farms where antimicrobials are used for therapeutic and growth promotion purposes. Another important facet of the swine industry is the interest in the antimicrobial free (ABF) production systems. The projected increase of 20-30% in the consumption of food products reared under antimicrobial free conditions in the United States necessitates the need to determine the status of major foodborne pathogens such as Campylobacter in swine reared under the ABF system. The purpose of this study was to determine the prevalence, antimicrobial resistance and the genetic diversity of Campylobacter isolated from swine reared in the two production systems at preharvest and during slaughter process. To better understand the epidemiology of this pathogen in the ABF system, similar comparisons were made between ABF herds reared in intensive units barns to those reared in the extensive environment. C. coli was the predominant species (99%) isolated from both the systems. High prevalence observed at the nursery level indicates that sows could act as an important source of transmission. The role played by environmental factors in transmission was highlighted by higher prevalence observed in ABF pigs reared outside with free access to soil and water. Significant reduction in prevalence was seen at the slaughter level though C. coli were isolated from post-chill samples. We recommend adding an intervention step after chilling and before packaging of the pork product. Highest frequency of resistance was seen against tetracycline and erythromycin in both the systems. We found significant association between antimicrobial resistance and the conventional production system. High frequency of pansusceptible isolates from the ABF system could be due to the absence of selective pressure of antimicrobials. Resistance against the fluoroquinolone ciprofloxacin and multidrug resistant C. coli in both systems points to the role of other unknown selective pressures and /or risk factors besides antimicrobial use. This is the first study in the US reporting ciprofloxacin resistant C. coli from swine. Ciprofloxacin resistance in C. coli is concerning as this antimicrobial is used for treating severe cases of campylobacteriosis in humans. Multilocus sequence typing was found to be a better method compared to pulsed field gel electrophoresis for genotyping C. coli based on its discriminatory power and throughput. High genetic diversity of C. coli was observed in the swine production systems as indicated by the weak clonal structure of the isolates tested. Evidence of a lineage in the ABF system will help in comparing C. coli isolates from different parts of the world. Linkage equilibrium between the housekeeping genes in isolates from the two systems could explain the high antimicrobial resistance exhibited by ABF pigs irrespective of use of antimicrobials at the farm. Overall, this study highlights the dissemination of antimicrobial resistant C. coli in swine irrespective of the production system. Resistance detected in the absence of antimicrobial selective pressure highlights the role played by unknown factors that need to be determined. We recommend studies that should be focussed on finding these sources so that effective preharvest control measures could be implemented. Finally, we recommend genotyping more isolates to better understand the epidemiology of C. coli in the swine environment.
The role of molecular methods in the detection of pathogens in food
(2004-03-18) Stevens, Kelly A.; Lee-Ann Jaykus, Committee Chair; Wondwossen Gebreyes, Committee Member; Craig Altier, Committee Member; MaryAnne Drake, Committee Member
The research described here addresses several issues associated with testing food samples for the presence of pathogens. Based on three different perspectives, the following major objectives are noted: (i) the use of molecular amplification methods to detect Listeria monocytogenes and Salmonella enterica serovar enteritidis directly from food samples, bypassing the need for cultural enrichment; (ii) evaluation of automated ribotyping for the differentiation of Salmonella enterica serovar Typhimurium strains; and (iii) development of a decision model to evaluate a pathogen testing decision in industry. In the first study, we developed and evaluated a method for the direct detection of foodborne pathogens without prior cultural enrichment. Eleven-gram samples of plain nonfat yogurt or mild cheddar cheese were seeded with L. monocytogenes or S. enterica Enteritidis at levels of 10, ², - 10, ⁶, CFU per sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9,700 x g) followed by detection using both cultural and molecular methods. Molecular detection limits of 10, ³,and 10, ¹, CFU per 11 g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. In a second and related study, alternative nucleic acid preparation methods were evaluated to improve the direct detection of Listeria monocytogenes from a frankfurter matrix. Using a combined concentration / extraction sample preparation, 11-g samples were concentrated 100-fold to 100 μl with recovery of target nucleic acids which were further purified by column chromatography and specific bacterial rRNA isolation using two magnetic bead-based technologies, i.e., MICROBEnrich® and MICROBExpress®. PCR detection limits were 10, ⁵, CFU/11g sample and RT-PCR detection limits were 10, ³, CFU/11g. Detection limits were improved an additional 10-fold (to 102 CFU/11g) when extracted RNA was further purified using MICROBExpress®. The third study evaluated the RiboPrinter®l microbial characterization unit for its ability to differentiate thirty-nine isolates of multi-drug resistant Salmonella enterica serovar. Typhimurium. Phenotypically, the isolates varied by phage type and marginally by antibiotic resistance pattern. However, the strains could not be meaningfully differentiated from one another using ribotyping, suggesting that in this case, phenotypic methods may be more discriminatory than this molecular typing method. In the final study, decision analysis tools were used to develop a model addressing the issues encountered when making a testing decision. From a food processors perspective, three potential consequences of foodborne pathogen contamination were considered as elements of the decision, e.g., no consequences, regulatory recall without disease, and disease outbreak. Accordingly, the inputs of the model were (i) costs associated with food-borne contamination (business and health related costs); (ii) reliability of testing; and (iii) prevalence of contamination. In general, the model indicated that testing for highly prevalent pathogens may provide an improvement in food safety but end product testing for pathogens of low prevalence should be carefully considered and may not be justified due to limited return on investment. These efforts represent continued progress in harnessing the power and diversity of molecular methods for the identification and characterization of foodborne pathogens. The development of systematic approaches to making testing decisions is also justified and needed by the industry. Taken together, these studies add to our understanding of the impediments to application of rapid methods for the detection of foodborne pathogens, and provide some solutions to facilitate the practical use of these methods in the future.