Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: With Listeria, our work has focused on molecular epidemiology studies and on analysis of resistance to disinfectants, antibiotics, phage and environmental stress (oxidative stress, exposure to heavy metals, cold and freeze-thaw stress). We have expanded our characterization of temperature-dependent phage resistance among Listeria monocytogenes isolates of a major epidemic clonal group, epidemic clone II. We have also completed a five-year analysis of the molecular epidemiology of Listeria monocytogenes strains provided to us from the Centers for Disease Control and Prevention. We have expanded the analysis of the virulence impact of the loss of glutathione synthase in Listeria, and have also collaborated with Dr. J. Guy at the school of Veterinary Medicine (NCSU) to employ the chick embryo model for analysis of Listeria virulence. We have completed our analysis of the prevalence of premature stop codons in the virulence determinant Internalin A among Listeria from the environment of food processing plants. In collaboration with C. Czuprynski (Univ. of Wisconsin) we have assessed the requirement for purine biosynthesis for virulence of Listeria in a mouse model. We have continued the analysis of the prevalence of heavy metal and disinfectant resistance genes among non-pathogenic Listeria from the turkey processing plant environment, and the ability of the genes to be disseminated via conjugation. We have assessed the impact of exposure to disinfectant on development of resistance to antibiotics (and vice versa) and have investigated novel genes on plasmids harboring disinfectant resistance genes in Listeria. With Campylobacter, we have assessed the ability of Campylobacter from different host animals (turkeys, swine, wild birds, chickens grown free-range in diversified farms) to colonize chickens, using the day-old chick model. We have continued the analysis of genome sequence data from four Campylobacter strains (three from turkeys, one from swine). We have investigated the molecular basis for drug resistance in the genome of these strains. We have continued our partnership with the Intestinal Health Survey program, with focus on monitoring Campylobacter prevalence and drug resistance in young turkey flocks grown conventionally in North and South Carolina. We have completed the analysis of data from 2001-2008 related to the impact of the ban of a major antibiotic (enrofloxacin) on antibiotic resistance of Campylobacter form turkeys. We have continued the analysis of the role of capsule on stress tolerance in Campylobacter. Our dissemination efforts to communities of interest have included publications in the peer-reviewed literature, student presentations in research conferences, presentation at a turkey industry meeting, presentation at a science cafe, and seminars by students and by the principal investigator. PARTICIPANTS: A total of 39 individuals participated in this project. This included the PI, PI's lab manager, collaborators, graduate and undergraduate students. A total of three Ph.D. Dissertations and two Masters thesis have been supported partially or completely by the grant. One Ph.D. dissertation has been completed. Progress is continuing on all others. Participating individuals were: S. Kathariou, PI; R. M. Siletzky, laboratory manager; D. Elhanafi, collaborator, NCSU ; J. Guy, collaborator, NCSU; R. Azizoglu, collaborator, NCSU; D. Carver, collaborator, NCSU; Jesse Grimes, collaborator, NCSU ; J. Olson, collaborator, NCSU; D. Smith, collaborator, NCSU; F. Arritt, collaborator, NCSU, D. Hanson, collaborator, NCSU; C. D'Lima, collaborator, FDA; Steven Foley, Collaborator, FDA; D. Drevets, collaborator, University of Oklahoma; C. Czyprynski, collaborator, Univ. of Wisconsin; N. Faith, collaborator, Univ. of Wisconsin; J. B. Luchansky, collaborator USDA-ERRC; R. Mandrell, collaborator, USDA-ARS; W. Miller, collaborator, USDA-ARS ; K. Sperry, collaborator, Dept of Health, NC; L. Wolff, collaborator, Dept of Health, NC; L. M. Graves, collaborator, CDC; P. Gerner-Smidt, collaborator, CDC; R. Sahaghian, graduate student; S. White, graduate student; S. Lee, graduate student; D. Bautista, graduate student; I. Sybirtseva, graduate student; V. Dutta, graduate student; S. Ratani, graduate student; M. Islam, post-graduate researcher; D. Britt, graduate student; S. Katharios- Lanwermeyer, research intern, Michigan State University ; K. Casulli, undergraduate research intern; A. Kirby, undergraduate research intern; .N. Butterbaugh, undergraduate research intern ; Lola Crespo Rodrigues, visiting scientist, University of Vigo, Spain ; Mira Rakic-Martinez, visiting scientist, Belgrade university, Serbia. Extensive opportunities were provided for training and professional development for students and other participants in the project. Students presented their research at the Kathariou laboratory meetings, and were encouraged and guided to present their findings at local regional and international meetings. They received safety training and acquired expertise in safe laboratory procedures during work with potential human pathogens. They worked as members of teams, thus developing team-working skills. They were guided in the process of preparing manuscripts, theses and dissertations, and presentations (posters, oral presentations). They were guided in the critical analysis of relevant scientific literature. They were guided in becoming familiar with principles of research and professional ethics through continuous feedback and guidance, specific presentations, and attendance of relevant classes at the University. Assistance and guidance were provided in identifying professional, training and employment opportunities upon completion of their involvement in the laboratory. TARGET AUDIENCES: The findings are targeted to the food safety and public health sector, who would be interested in the identification and characterization of special attributes associated with Listeria and Campylobacter strains that compromise the safety of the food supply; to the food industry and environmental sanitation sector; to regulatory agencies and biotechnology sectors, since strains constructed in this work can optimize the use of phages as biocontrol for Listeria, and minimize the potential that phage will inadvertently select for especially problematic strains (e.g. Epidemic Clone II); to public health and regulatory agencies in need of microbiological data to assess the use of antimicrobials in animal production; to the animal industry sector (producers of poultry, swine, cattle), who would be interested in host animal-specific colonization with campylobacters, and in possible barriers to colonization stemming from host adaptations. The findings are also targeted to the microbiology, evolutionary biology and food safety communities, since they refer to fundamental adaptations of bacterial foodborne pathogens relevant to processes responsible for contamination of foods and ultimately for foodborne disease. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With Listeria, we elucidated aspects of resistance to quaternary ammonium disinfectants. Disinfectant resistance genes were induced by disinfectant and were expressed at higher levels at low temperatures than at 37C. Phage resistance genes of epidemic clone II were also characterized and shown to be induced at low temperature. Analysis of serotype 4b strains of Listeria from human listeriosis in a five-year period revealed three major clonal groups which have also been implicated in outbreaks of listeriosis. Several novel clonal groups were identified, one seemingly unique to North Carolina. A significant portion of Listeria strains of serotypes 1/2a and 1/2b from the environment of turkey processing plants harbored premature stop codons in the gene for Internalin A, suggesting that they have attenuated virulence and would make limited contributions to human foodborne listeriosis. Our data suggest that attenuated-virulence clones of these serotypes have populated the processing plant environment. However, serotype 4b strains, without exception, harbored intact Internalin A genes, suggesting that such strains likely have no defects in internalin A-associated virulence. A large fraction of strains of non-pathogenic Listeria species (L. welshimeri and L. innocua) from the turkey processing plants harbored resistance to cadmium and to quaternary ammonium disinfectants, and such resistance could be transferred via conjugation; non-pathogenic Listeria may serve as reservoir for such genes in food processing environments. With Campylobacter, we have identified a number of host-associated genes through analysis of genome sequence data and testing a panel of strains from diverse hosts through DNA-DNA hybridizations. A unique methylation of the genome was detected among swine-derived strains but not among strains from turkeys. We identified drug resistance genes on the chromosome and on the plasmids of the sequenced genomes. We showed that strains from diverse hosts were capable of colonization of chickens in the day-old chick model. Through our work with the Intestinal Health Survey program we have discovered major shifts in colonization and drug resistance profiles among Campylobacter from young turkey flocks grown conventionally in North and South Carolina. We have completed pilot studies of free-range chickens grown in a diversified commercial farm in N. Carolina, and showed that Campylobacter strains from these flocks were largely susceptible to antibiotics, suggesting that management style has pronounced impact on drug resistance in the Campylobacter strains that infect the birds. Our analysis of fluoroquinolone resistance data (2001-2008) revealed that the enrofloxacin ban did not have significant impact in reducing prevalence of resistance during this period. Instead, multidrug resistance (which includes resistance to fluoroquinolones) appeared to have been significantly increased post-ban. Our analysis of capsule-deficient mutants revealed that capsule may be essential for colonization of chicks, but not for environmental survival.
Publications
- Elhanafi D, V. Dutta, and S. Kathariou. 2010. Genetic characterization of plasmid-associated benzalkonium chloride resistance determinants in a Listeria monocytogenes strain from the 1998-1999 outbreak. Appl. Environ. Microbiol. 76:8231-8238.
- Wright, S., S. Wilson, W. G. Miller, R. E. Mandrell, R. M. Siletzky, and S. Kathariou. 2010. Differences in methylation at GATC sites in genomic DNA of Campylobacter coli from turkeys and swine. Appl. Environ. Microbiol. 76:7314-7317.
- Azizoglu, R. O., and S. Kathariou. 2010. Temperature-dependent requirement for catalase in aerobic growth of Listeria monocytogenes F2365. Appl. Environ. Microbiol. 76:6998-7003.
- Yildirim, S., D. Elhanafi, W. Lin, A. D. Hitchins, R. M. Siletzky, and S. Kathariou. 2010. Conserved genomic localization and sequence content of Sau3AI-like restriction-modification gene cassettes between Listeria monocytogenes Epidemic Clone I and selected strains of serotype 1/2a. Appl. Environ. Microbiol. 76:5577-5584.
- Azizoglu, R. O., and S. Kathariou. 2010. Impact of growth temperature and agar versus liquid media on freeze-thaw tolerance of Yersinia enterocolitica. Foodborne Pathog Dis. 7:1125-1128.
- Azizoglu, R. O., and S. Kathariou. 2010. Inactivation of a cold-induced putative RNA helicase gene of Listeria monocytogenes is accompanied by failure to grow at low temperatures but does not affect freeze-thaw tolerance. J. Food Prot. 73:1474-1479.
- Cheng, Y., J. W. Kim, S. Lee, R. M. Siletzky, and S. Kathariou. 2010. DNA probes for unambiguous identification of Listeria monocytogenes Epidemic Clone II strains. Appl. Environ. Microbiol. 76:3061-3068.
- Casulli, K., R. M. Siletzky, and S. Kathariou. 2010. Vector construction for generation of kpsM mutants in multidrug resistant Campylobacter strains. The 10th Annual NC State University Undergraduate Summer Research Symposium, Raleigh, NC.
- D. Lima, C., and S. Kathariou. 2010. Stability of multidrug resistance among Campylobacter coli from turkeys. International Association for Food Protection Annual Meeting.
- Dutta, V., D. Elhanafi and S. Kathariou. 2010. Temperature-dependent regulation of a benzalkonium chloride resistance determinant in Listeria monocytogenes. American Society for Microbiology General Meeting.
- Dutta, V., D. Elhanafi and S. Kathariou. 2010. Regulation of plasmid- associated benzalkonium chloride resistance determinants in an outbreak strain of Listeria monocytogenes. Sigma Xi Annual Meeting & International Research Conference, November 12-13, 2010, Raleigh, NC.
- Dutta, V., D. Elhanafi, M. Rakic-Martinez, J.-W. Kim, R. M. Siletzky and S. Kathariou. 2010. Unintended consequences of pathogen control efforts in intensive food production and processing systems. Sigma Xi Annual Meeting & International Research Conference, November 12-13, 2010, Raleigh, NC.
- Faith, N. G., J.-W. Kim, S. Kathariou, R.Sahaghian, J. B. Luchansky and C. Czuprunski. 2010. Reduced virulence of an adenylosuccinate lyase transposon mutant of a serotype 4b strain of Listeria monocytogenes. International Symposium on Problems of Listeriosis (ISOPOL).
- Katharios-Lanwerneyer, S. G., S. Kathariou, and J. M. Tiedje. 2010. Transfer and stability of cadmium resistance among Listeria welshimeri and L. innocua strains from turkey processing plants. American Society for Microbiology General Meeting.
- Kathariou, S. 2010. Listeria monocytogenes as a paradigm for environmental adaptations in foodborne pathogens: lessons and implications. Korea Microbiology & Biotechnology Conference, Seoul S. Korea.
- Kathariou, S. 2010. Environmental adaptations in foodborne pathogens: focus on Listeria, disinfectants and antibiotics. XXXIII International Congress οf τhe Society for Microbial Ecology in Health and Disease.
- Kathariou, S., J. Olson, E. Altermann, and R. M. Siletzky. 2010. Genomic attributes associated with host adaptation in Campylobacter jejuni and Campylobacter coli from poultry and swine. USDA Annual Principal Investigators Meeting.
- Lee, S., and S. Kathariou. 2010. Two novel restriction-modification systems in Listeria monocytogenes. American Society for Microbiology General Meeting.
- Lee, S., and S. Kathariou. 2010. Novel restriction-modification systems in Listeria monocytogenes. North Carolina regional ASM meeting, Greenville, NC.
- Lok, M., Y. Chen. B. Verghese, P. Evans, S. Kathariou, and S. Knabel. 2010. Molecular subtyping of Listeria monocytogenes Epidemic Clones II and III by sequence analysis of the upstream and downstream comk prophage junction fragments. International Association for Food Protection Annual Meeting.
- Rahimi, S., J.L. Grimes, S. Kathariou, and R.M. Siletzky. 2010. Reduction of Clostridium perfringens colonization in turkey poults by feeding Primalac. Poultry Science Association Annual Conference.
- Sybirtseva, I., F. M. Arritt, S. Kathariou, D. J. Hanson, D. P. Smith, K. G. Martino, J. B. Luchansky. 2010. Efficacy of oxalic acid to reduce Salmonella spp. at various stages of poultry processing. International Poultry Scientific Forum, Atlanta, GA, January 25, 2010.
- Sybirtseva, I., S. Kathariou, D. J. Hanson, D. P. Smith, J. B. Luchansky, K. G. Martino, and F. M. Arritt. 2010. Efficacy of fumaric acid to Reduce Salmonella spp. at various stages of poultry processing. International Association for Food Protection annual meeting.
- Siletzky, R. M., D. Carver, S. Wright, M. Islam, C. D.Lima, H. Ahmad and S. Kathariou. 2010. Prevalence and drug resistance in Campylobacter from turkeys. Turkey Days Annual Conference, Wilmington, NC.
- Sybirtseva, I., S. Kathariou, D. J. Hanson, D. P. Smith, J. B. Luchansky, K. G. Martino, and F. M. Arritt. 2010. Are three replications from a single lot adequate to assess antimicrobial efficacy? American Meat Science Association 63rd Reciprocal Meat Conference, Texas Tech University, Lubbock, TX, June 20-23, 2010.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: With Listeria, our work has included the analysis of heavy metal resistance and resistance to the quaternary ammonium disinfectant, benzalkonium chloride among isolates from environmental, food and clinical sources. This analysis has included the study of the impact of temperature and exposure to the corresponding agents on the expression of genes involved in resistance. We have assessed temperature-dependent phage resistance among Listeria monocytogenes isolates of a major epidemic clonal group, epidemic clone II. We have also pursued the molecular epidemiology of Listeria monocytogenes strains provided to us from the Centers for Disease Control and Prevention in order to assess the prevalence of distinct clonal groups in human illness. We have determined the potential of clone-specific DNA fragments as probes that be used to specifically identify strains of major epidemic clones. We have determined the impact of cold stress and cold tolerance on the ability of Listeria to tolerate subsequent stress, such as repeated freezing and thawing (cryotolerance). We have also determined whether genes mediating responses to oxidative stress in Listeria are involved in cryotolerance. We have determined the ability of Listeria to survive and grow in the cold in raw and pasteurized milk. We have analyzed the prevalence of a specific virulence attenuation marker among Listeria from the environment of food processing plants. We have also examined the prevalence of heavy metal and disinfectant resistance genes among non-pathogenic Listeria from the turkey processing plant environments, and the ability of the genes to be disseminated via conjugation. With Campylobacter, we have assessed the ability of Campylobacter from different host animals (turkeys, swine) to colonize birds using the day-old chick model. We have determined the genome sequence of four Campylobacter strains (one from swine, three from turkeys) and have pursued the analysis of the sequence data. We have continued our surveillance of Campylobacter prevalence and drug resistance in young turkey flocks grown conventionally in North and South Carolina. We have determined the impact of the ban of a major antibiotic (enrofloxacin) on resistance of Campylobacter form turkeys to this antibiotic, including in our analysis data from 2001 to the present. We have determined the ability of transformation (uptake of naked DNA by cells) to transfer resistance to tetracycline and to fluoroquinolones among strains from different animal hosts and with different genotypes. We have assessed the ability of Campylobacter to survive in raw and pasteurized milk at low temperature. We determined the impact of the bacterial capsule on the ability of Campylobacter to colonize chickens, to survive in milk and in water, and to tolerate environmental stresses. Our dissemination efforts to communities of interest have included publications in the peer-reviewed literature, student presentations in research conferences, one town hall style meeting, and seminars by students and by the principal investigator. PARTICIPANTS: A total of 29 individuals participated in this project. This included the PI, PI's lab manager, collaborators, graduate and undergraduate students. A total of four Ph.D. Dissertations and four Masters thesis have been supported partially or completely by the grant. One Ph.D. dissertation and three M.Sc. thesis (N. Carolina State University) have been completed. Progress is continuing on all others. Participating individuals were: S. Kathariou, PI; C. Czyprynski, collaborator, Univ. of Wisconsin; N. Faith, collaborator, Univ. of Wisconsin; J. B. Luchansky, collaborator USDA-ERRC; K. Sperry, collaborator, Dept of Health, NC; L. Wolff, collaborator, Dept of Health, NC; L. M. Graves, collaborator, CDC; B. Swaminathan, collaborator, CDC; P. Gerner-Smidt, collaborator, CDC; R. M. Siletzky, laboratory manager; D. Elhanafi, NCSU collaborator; B. Chapman, NCSU collaborator; D. Buchwalter, NCSU collaborator; , R. Sahaghian, graduate student; S. Mullapudi, graduate student; R. Azizoglu, graduate student; S. White, graduate student; S. Lee, graduate student; V. Dutta, graduate student; M. Islam, graduate student; D. Britt, graduate student; J. Xiong, graduate student; M. Lechner, undergraduate student ; M. Yonas, undergraduate student; S. Ratani, undergraduate student; S. Lanwermeyer, undergraduate student; W. Jiang, undergraduate student; Erin Whitley, undergraduate student; J. Beane, undergraduate student. Extensive opportunities were provided for training and professional development for student and other participants in the project. Students presented their research at the Kathariou laboratory meetings, and were encouraged and guided to present their findings at local (University and state) and international meetings. They received safety training and acquired expertise in safe laboratory procedures during work with potential human pathogens. They worked as members of teams, thus developing team-working skills. They were guided in the process of preparing manuscripts, theses, and presentations (posters, oral presentations). They were guided in the critical analysis of relevant scientific literature. They were guided in becoming familiar with principles of research and professional ethics through continuous feedback and guidance, specific presentations, and attendance of relevant classes at the University. Assistance and guidance were provided in identifying professional, training and employment opportunities upon completion of their involvement in the laboratory. TARGET AUDIENCES: The findings are targeted to the food safety and public health sector, who would be interested in the identification and characterization of special attributes associated with Listeria and Campylobacter strains that compromise the safety of the food supply; the food industry and environmental sanitation sector; to regulatory agencies and biotechnology sectors, since strains constructed in this work can optimize the use of phages as biocontrol for Listeria, and minimize the potential that phage will inadvertently select for especially problematic strains (e.g. Epidemic Clone II); to public health and regulatory agencies in need of microbiological data to assess timely and controversial issues such as raw milk consumption and the use of antimicrobials in animal production; to the animal industry sector (producers of poultry, swine, cattle), who would be interested in host animal-specific colonization with campylobacters, and in possible barriers to colonization stemming from host adaptations. The findings are also targeted to the microbiology, evolutionary biology and food safety communities, since they refer to fundamental adaptations of bacterial foodborne pathogens with zoonotic potential, and are directly relevant to processes involved in contamination of foods and ultimately in foodborne disease. PROJECT MODIFICATIONS: None; work has proceeded as planned.
Impacts
With Listeria, we have discovered that heavy metal resistance and resistance to the quaternary ammonium disinfectant, benzalkonium chloride (BC) was highly prevalent among isolates from the environment of turkey processing plants. All BC-resistant isolates were also resistant to cadmium. A novel cadmium resistance determinant, first detected in pLM80 from the 1998-1999 outbreak, was found significantly more frequently among isolates that were both BC and cadmium resistant than among those resistant to cadmium but susceptible to BC. Furthermore, non-pathogenic Listeria spp. also frequently harbored BC and cadmium resistance, as well as this novel cadmium-resistance determinant. We identified a locus responsible for temperature-dependent resistance of epidemic clone II (ECII) strains (i.e. the clone that includes the serotype 4b strains responsible for two multistate outbreaks of listeriosis, in 1998-1999 and in 2002) to listeriaphage. Expression of a key gene in this locus were found to be dependent on temperature, being induced at low temperature. This locus was one of two that we identified to be 100% specific for ECII, and absent from all other tested serotype 4b strains. By using such probes we have assessed the clonal composition of serotype 4b strains randomly selected from different states in the nation, and provided to us by the Centers for Disease Control and Prevention. Our findings of a large panel of serotype 4b strains from 2003-2008 showed that epidemic clones I and II remained major contributors to human listeriosis in the US, but also identified several novel clonal groups being further characterized in our laboratory. Unlike other cold-tolerant bacteria, Listeria was found to be much more tolerant to freeze-thaw stress when grown at temperatures encountered during infection (37C) than in the cold. We found that expression of cold-essential genes (such as a cold-induced gene encoding an RNA helicase) was not required for cryotolerance. With Campylobacter, we found that swine-adapted strains can colonize the day-old chick model efficiently. We found evidence for host preference in the genome sequences of strains from different meat animal hosts (turkeys, swine, cattle, chicken). We found that Campylobacter continues to be highly prevalent among turkeys grown conventionally in North and South Carolina, and have assessed the impact of different integrators in prevalence and drug resistance. We noted increases in prevalence of fluoroquinolone resistance post-ban, as well as prevalence of multidrug resistant (MDR) strains. Several distinct strain types were detected among MDR C. jejuni and C. coli from turkeys. We found pronounced strain influence on survival of Campylobacter in raw and pasteurized milk. Capsule of the bacteria was found essential for colonization of birds. Campylobacter showed pronounced ability to transfer resistance to tetracycline via transformation, with efficiency depending on the source and the genotype of the strain.
Publications
- Mullapudi, S., R. M. Siletzky, and S. Kathariou. 2009. Diverse cadmium resistance determinants in Listeria monocytogenes from the environment of turkey processing plants. Appl Environ Microbiol. Nov 30. [Epub ahead of print]
- Pan, Y, F. Breidt, Jr, and S. Kathariou. 2009. Competition of Listeria monocytogenes serotype 1/2a and 4b strains in mixed culture biofilms. Appl. Environ. Microbiol. 75:5846-5852.
- Vishnivetskaya, T. A., S. Kathariou, and J. M. Tiedje. 2009. The Exiguobacterium genus: biodiversity and biogeography. Extremophiles. 13:541-555.
- Kim, J. W., and S. Kathariou. 2009. Temperature-dependent phage resistance of Listeria monocytogenes epidemic clone II. Appl. Environ. Microbiol. 75:2433-2438.
- Azizoglu, R. O., J. Osborne, S. Wilson, and S. Kathariou. 2009. Role of growth temperature on freeze-thaw tolerance of Listeria spp. Appl. Environ. Microbiol. 75:5315-5320.
- Gu, W., R. S. Siletzky, S. Wright, M. Islam, and S. Kathariou. 2009. Antimicrobial susceptibility profiles and strain type diversity of Campylobacter jejuni isolates from turkeys in eastern North Carolina. Appl. Environ. Microbiol. 75:474-482.
- Ratani, S. S., V. Dutta, R. O. Azizoglu, and S. Kathariou. 2009. Characterization of a copper resistance determinant in Listeria monocytogenes using a mariner-based transposition system. American Society for Microbiology NC Branch.
- Beane, J. B., V. Dutta, and S. Kathariou. 2009. Does the Ciliated Protozoan Tetrahymena Influence the Survival of the Food-borne Pathogen Listeria monocytogenes The 8th Annual NC State University Undergraduate Summer Research Symposium, Raleigh, NC.
- Jiang, W., R. M. Siletzky, and S. Kathariou. 2009. Prevalence and Clone Stability in Multidrug Resistant Campylobacter coli from Turkeys in North Carolina. The 8th Annual NC State University Undergraduate Summer Research Symposium, Raleigh, NC.
- Britt, D. and S. Kathariou. 2009. Phenotypic characterization of capsule-deficient mutants of various Campylobacter strains. 2009 Molecular Biotechnology Research Symposium, Raleigh, NC
- Whitley, E., D. Clare, R. Siletzky, and S. Kathariou. 2009. Antibacterial Properties of Milk Components in Campylobacter jejuni. The 9th Annual NC State University Undergraduate Summer Research Symposium, Raleigh, NC
- Azizoglu, R. O. 2009. Temperature-dependent freeze-thaw tolerance and genetic characterization of stress response mechanisms of Listeria. Ph.D. dissertation, North Carolina State University.
- Faith, N. G., J.-W. Kim, S. Kathariou, R. Sahaghian, J. B. Luchansky and C. Czuprunski. 2009. Virulence for mice, resistance to synthetic gastric fluid and biofilm formation of a strain of Listeria monocytogenes serotype 4b isolated from an listeriosis outbreak associated with hot dogs. International Association for Food Protection Conference, Grapevine, TX.
- Xiong, J. 2009. Survival of Animal-derived Campylobacter Strains in Raw and Pasteurized Milk, and the Roles of Capsule in Campylobacter Survival in vitro, and in Chick Colonization. M. Sc. Thesis, North Carolina State University.
- Islam, M. 2009. Prevalence and Dissemination of Antimicrobial Resistance among Campylobacter jejuni and Campylobacter coli from Meat-Animals. M. Sc. Thesis, North Carolina State University.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Research has proceeded as planned on both Listeria and Campylobacter. With Listeria, we have characterized a library of strains from the turkey processing plant environment in regard to their resistance to disinfectant and to heavy metals. We have identified genes responsible for resistance to BC on the large plasmid harbored by certain strains. The prototype for this plasmid is pLM80, identified during the genome sequencing of a strain implicated in the 1998-99 outbreak of listeriosis traced to contaminated hot dogs. The genes for cadmium resistance were also localized on plasmids. Three different cadmium resistance gene cassettes have been identified in Listeria. We have also analyzed the strains from the turkey processing plants in terms of the prevalence of mutated internalin A genes, which harbor premature stop codons. The resistance of strains form the processing plant environments to Listeria specific phages has also been determined. Phages were isolated from environmental samples derived from the turkey processing plants, and screened in terms of their ability to infect and kill Listeria strains from these environments. Strains from major outbreaks were extensively characterized in terms of their susceptibility to phage, to determine the suitability of phage as possible biocontrol for such strains. The impact of growth conditions on freeze thawing tolerance of Listeria was also investigated. With campylobacter, we have continued analysis of prevalence of Campylobacter jejuni and Campylobacter coli among turkeys grown commercially in north and South Carolina. We also have assessed prevalence of resistance to a panel of antibiotics, with special emphasis on resistance to fluoroquinolones. We have investigated genetic diversity, population structure and antimicrobial resistance of Campylobacter from turkeys and other meat animals (hogs, cattle). To investigate the possible role of capsule in Campylobacter physiology, environmental adaptations, and ability to colonize poultry, we constructed mutants in a capsule biosynthesis gene. We successfully performed chicken colonization assays using two Campylobacter coli strains, of turkey and hog origin, respectively. We have assessed the relative ability of Campylobacter jejuni, Campylobacter coli, and Listeria to survive in raw versus pasteurized milk. PARTICIPANTS: The project provided numerous opportunities for training in bacteriology, bacterial genetics and physiology, subtyping and determinations of susceptibility to antimicrobials, disinfectant, and heavy metals for twelve graduate students (Sandra Wright, Mohammed Islam, Jiajuan Xiong, Kamfai Chan, Joo-Sung Kim, Jae-Won Kim, Cheng Ying, Savitri Mullapudi, Sangmi Lee, Reha Azizoglu, Sheea White, Sandra Wright); two postdoctoral scientists (Weimin Gu, Driss Elhanafi); four undergraduate research interns (Julie Darly, Simone Wilson, Jach Byrd, Clara Tang); eleven collaborating scientists (M. Morrow and Donna Carver, North Carolina State University, Dept of Animal Science and Dept of Poultry Science, respectively; Nan Faith and Chuck Czuprynski, University of Wisconsin; Nattawan Promadej, university of Hawaii, currently at the Centers for Disease Control and Prevention; Q. Zhang and J. Luchansky, USDA-ARS; Brien Nudeck, University of Tennessee; Kate Sperry, Leslie Wolf and Justin Edwards, North Carolina Department of Health, Raleigh, NC); one research laboratorian (S. Romine); one visiting scientist (Natalia Markelova, Moscow State university, Russia). In addition, the laboratory manager, Robin Siletzky and the PI Sophia Kathariou participated in the project. TARGET AUDIENCES: Target audiences for the research project and findings thereof include individuals associated with the turkey and meat production industry, who will be interested in knowing the prevalence of disinfectant and phage resistance among Listeria strains from the environment of the processing plants. These individuals would also be interested in Campylobacter prevalence and subtypes in meat animals pre-harvest, the possible overlap with strains from human illness, and the antimicrobial susceptibility attributes of the organisms. A second target audience would include individuals from regulatory agencies, e.g. agencies approving and monitoring use of phage for Listeria biocontrol, and stakeholders with animal health interests and responsibilities, who would be especially interested in the findings related to the impact of the fluoroquinolone ban on fluoroquinolone resistance in campylobacter form turkeys. A third target audience would include members of the scientific community, who would be interested in the population structure and genomic attributes of the organisms, the implications of the findings for the pathogens' evolution and environmental adaptations, the genetic basis for host and niche adaptation, and horizontal gene transfer of resistance determinants in Listeria and Campylobacter. A fourth target audience would be members of the food safety community, who would be interested in multiple aspects of the findings related to the adaptations of the pathogens and their survival in the processing plants and in specific commodities (e.g. raw milk); Listeria and Campylobacter are associated with significant public health and socioeconomic burden in the United States and other industrialized nations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of disinfectant resistance among Listeria strains from the environment of turkey processing plants revealed that all strains resistant to the quaternary ammonium compound benzalconium chloride (BC) were also resistant to the heavy metal cadmium. A novel finding was that strains resistant both to BC and to cadmium had a higher likelihood of harboring one specific cadmium resistance cassette than strains which were only resistant to cadmium, but susceptible to BC. A number of the strains harbored internalin A genes with premature stop codons. Sequence analysis provided accurate information of the prevalence of the different stop cordon mutations among these strains. A high proportion of strains of serotype 1/2a and 1/2b were found resistant to wide host range phages isolated from the processing plants, as well as to other tested wide host ranges. In contrast, strains of serotype 4b were largely phage susceptible. Strains from one of the major epidemic clones were uniformly resistant to the phages under certain growth conditions, suggesting that resistance would be likely in the environment of the processing plants and in foods. These findings raise suggest great caution in possible uses of phage as biocontrol: such use might eliminate other strains, while leaving problematic epidemic strains unharmed. The genetic basis of phage resistance among the epidemic strains was investigated, and a novel gene cassette was identified using transposon mutagenesis. Analysis of freeze thawing tolerance following growth under different conditions revealed that cryotolerance was significantly enhanced following growth at 37C, a temperature that also permits virulence gene expression in Listeria. Fluoroquinole resistance of turkey-derived Campylobacter has continued to be highly prevalent, in spite of the 2005 ban for fluoroquinolone use in poultry. The findings suggest that the resistant strains are stable, and raise the possibility that other conditions in the turkey industry (e.g. treatment with other antibiotics that selects for multidrug resistant strains that had acquired resistance to fluoroquinolones prior to the ban) may inadvertently exert pressure for resistance. The findings also suggest the need for continued sampling, to adequately obtain data related to the time required to detect a possible impact of the ban. Strain subtyping revealed that strains from turkeys constituted a number of well-defined clonal groups, and also suggested that human strains were genetically similar to strains from one of the turkey-derived clones, as well as to clonal groups from cattle. Chicken colonization assays suggested differences in relative colonization efficiency among strains. Survival of Campylobacter was found to be generally more pronounced in pasteurized milk than in raw milk, with pronounced differences detected among strains. In contrast, Listeria survived and actually grew both in raw and pasteurized milk. The raw milk studies provide urgently needed data to adequately inform consumers on possible food safety hazards associated with this commodity.
Publications
- Gu, W., Islam, M., Siletzky, R., Kathariou, S. (2008). Prevalence of ciprofloxacin resistance in thermophilic campylobacters from turkeys in N. Carolina before and after the fluoroquinolone ban. American Society for Microbiology General Meeting (P-054).
- Faith, N., Kathariou, S., Cheng, Y., Promadej, N., Neudeck, B. L. , Zhang, Q., Luchansky, J., Czuprynski, C. (2008). The role of L. monocytogenes serotype 4b gtcA in gastrointestinal listeriosis in A/J mice. Foodborne Pathog Dis. Nov 9. [Epub ahead of print]
- Kim, J. W., Siletzky, R. M., Kathariou, S. (2008). Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States. Appl Environ Microbiol. 74:6623-30.
- Sperry, K. E. , Kathariou, S., Edwards J. S. , and Wolf, L. A. (2008). Multiple-locus variable-number tandem-repeat analysis as a tool for subtyping Listeria monocytogenes strains. J. Clin. Microbiol. 46:1435-1450.
- Mullapudi, S., Siletzky, R. M., and Kathariou, S. (2008). Heavy metal and benzalkonium chloride resistance of Listeria monocytogenes from the environment of turkey processing plants. Appl. Environ. Microbiol. 74:1464-1468.
- Cheng, Y., Promadej, N., Kim, J. W., and Kathariou, S. (2008). Teichoic acid glycosylation mediated by gtcA is required for phage adsorption and susceptibility of Listeria monocytogenes serotype 4b. Appl. Environ. Microbiol. 74:1653-1655. Jan 11; [Epub ahead of print]
- Cheng, Y., Siletzky, R. M., and Kathariou, S. 2008. Genomic divisions / lineages, epidemic clones and population structure of Listeria monocytogenes. Handbook of Listeria monocytogenes. Dongyou Liu ( Ed), Taylor & Francis Group, Inc.
- Gu W., Siletzky R.M., Wright S., Islam M., Kathariou S. (2008) Antimicrobial susceptibility profiles and strain type diversity of Campylobacter jejuni from turkeys produced in eastern North Carolina. Appl Environ Microbiol. Nov 21. [Epub ahead of print]
- Wright S. L., Carver D. K., Siletzky R. M., Romine S., Morrow W. E., Kathariou S. (2008) Longitudinal study of prevalence of Campylobacter jejuni and Campylobacter coli from turkeys and swine grown in close proximity. J Food Prot. 71:1791-1796.
- Kim J. S., Kim J. W., Kathariou S. (2008). Differential effects of temperature on natural transformation to erythromycin and nalidixic acid resistance in Campylobacter coli. Appl Environ Microbiol. 74:6121-6125
- Chan K. Elhanafi D. Kathariou S. (2008). Genomic evidence for interspecies acquisition of chromosomal DNA from Campylobacter jejuni by Campylobacter coli strains of a turkey-associated clonal group (cluster II). Foodborne Pathog Dis. 5:387-38.
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