Browsing by Author "Dr. Sophia Kathariou, Committee Chair"

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Genetic Characterization of Genes Specific to Listeria monocytogenes Epidemic-Associated Serotype 4b Strains
(2007-04-08) Cheng, Ying; Dr. Hosni Hassan, Committee Member; Dr. Sophia Kathariou, Committee Chair; Dr. Jonathan C. Allen, Committee Member; Dr. Craig Altier, Committee Member
Listeria monocytogenes, a food-borne pathogen of humans and animals, can cause severe listeriosis with relatively high mortality. A cluster of closely related strains of L. monocytogenes (designated Epidemic Clone I) have been implicated in numerous outbreaks in Europe and North America, including the California outbreak of 1985. L. monocytogenes strains implicated in the 1998-1999 and the 2002 multistate outbreaks in the USA represent a unique epidemic-associated clonal group, designated Epidemic Clone II (ECII). Comparative genomic analyses across five genomes from different L. monocytogenes isolates and Listeria species identified a genomic region (region-18) in serotype 4b strains that may have been acquired by horizontal gene transfer. Region-18 is either absent or markedly divergent in ECII strains but conserved among other serotype 4b strains. Region-18 is flanked by a large gene encoding a putative cell-wall associated protein (wap) on one side and a well-known virulence gene internalin A (inlA) on the other side in serotype 4b strains. PCR primers and DNA probes derived from this ECII-specific region-18 can readily differentiate ECII strains from other serotype 4b strains. This facilitates the detection and monitoring of these strains belonging to ECII clonal group in foods, clinical samples, and the environment. Genetic characterization of wap by the construction of deletion mutants suggested that the ECII wap mutant but not the ECI wap mutant may be involved in specific environmental adaptations such as surface adherence and possibly biofilm formation in ECII strains. Mutational and functional analyses showed that the deletion mutant of region-18 in ECII had an enhanced death rate during post-stationary incubation at 42℃,suggesting that the ECII-specific region-18 may be implicated in post-stationary stress responses.Two c72.44-negative variants of epidemic-associated L. monocytogenes serotype 4b strains were isolated from laboratory cultures. Naturally occurring c74.22-negative variants that exist under laboratory conditions without any noticeable phenotypic differences from their original forms may complicate the analysis of phage sensitivity and pathogenic characteristics.
A Longitudinal Study of Prevalence, Antibiotic Resistance and Strain Types of Campylobacter Isolates in Turkeys
(2005-11-21) Joshi, Shilpa; Dr. Fred Breidt, Committee Member; Dr. James Brown, Committee Member; Dr. Brian Sheldon, Committee Member; Dr. Sophia Kathariou, Committee Chair
Campylobacter has been recognized as a leading bacterial cause of human gastroenteritis in the United States, with ca. 2.4 million cases each year. Epidemiological data suggest that contaminated products of animal origin, especially raw poultry contributes significantly to campylobacteriosis with C. jejuni being the most prevalent species accounting for 90% of the infections. Research has been primarily focused on Salmonella in broilers and little information is available on colonization of Campylobacter in turkeys. Due to the growing popularity of turkey as a food commodity in the US, and North Carolina being a leading turkey producing state, it is important to focus attention on this zoonotic pathogen and its sources of colonization in turkey flocks. In an effort to address these issues, a longitudinal study was conducted on the prevalence, antibiotic resistance and strain types of Campylobacter isolates obtained from twelve turkey farms in Eastern North Carolina. Fecal samples were collected at 5, 7, 10, 13 and >13 weeks of age during the lifetime of the flock. Samples were tested for resistance to antibiotics following the National Committee for Clinical Laboratory Standards (NCCLS) guidelines and genotyping methods, including fla typing and Pulsed Field Gel Electrophoresis (PFGE) were employed to investigate the genetic diversity among these isolates. Farms involved in the study were chosen based on prevalence data for C. jejuni, which suggested high prevalence of this organism at different time points during the lifetime of the flock. The first study focused on C. jejuni isolates in a brooder/grow-out facility. It was of particular interest due to the trends seen in resistance of brooder isolates to fluoroquinolones (FQs) and persistence of a particular genotype in the brooder and the grow-outs. 92.3% of the C. jejuni isolates in house #1 (brooder) were sensitive to FQs while 50% of the isolates in house #2 (brooder) were resistant to FQs. TAK and TAKQ were the two main categories of antibiotic resistance seen in house #1 and #2 respectively. Genotyping showed the presence of one predominant fla and SmaI type in both the houses with a small fraction of isolates from the grow-out flocks having this strain type. These findings suggested that isolates of the same genotype may differ in FQ resistance and that diversity can be observed even within the same farm. The objectives of the second study were to determine the prevalence of Campylobacter in twelve turkey farms and antibiotic resistance patterns among the isolates in these farms. Even though our focus was on C. jejuni isolated from these farms, information about C. coli isolates was also obtained. Resistance of Campylobacter isolates to clinically relevant antibiotics such as erythromycin and FQs was substantially high with multi-resistance more commonly observed in C. coli. Almost 100% of the isolates were resistant to tetracycline and ampicillin. A mean of 28.2% C. jejuni isolates and mean of 63.4% C. coli isolates had a FQ MIC of >160 μg/ml. The third study evaluated the genetic diversity among Campylobacter isolates and determined whether particular strain types persist throughout the lifetime of the flock. It was found that 3 of the 8 brooder/grow-out pairs showed the presence of one persistent fla and PFGE type with certain strain types being found at more than one sampling timepoint. Genetic diversity was seen with 3-11 fla and 2-10 PFGE types in each flock. In 3 of the 6 brooder/grow-out pairs, C. jejuni strain types from the brooders were found in grow-outs. Genotyping techniques were effective in subtyping the isolates with a mean of 14.8 isolates per flock being subtyped (Total N=296). In conclusion, data collected in such research studies will contribute to further understand Campylobacter colonization in turkeys, especially in regards to elucidating prevalence, antibiotic resistance, and population diversity patterns of the pathogen in commercial turkey farms.
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
(2009-09-10) Xiong, Jiajuan; Dr. Sophia Kathariou, Committee Chair; Dr. Ilenys M. Perez-Diaz, Committee Member; Dr. Jonathan Olson, Committee Member
Campylobacter spp. is a major cause of acute bacterial gastroenteritis in the developed countries. It can survive in a wide range of environments, raw milk and poultry are major reservoirs. Survival of animal-derived Campylobacter strains, including six C. jejuni and six C. coli, was examined in raw and pasteurized milk at 4oC. Most (11/12) of the Campylobacter strains survived in both raw and pasteurized milk for at least 14 days. The most resistant strain, a C. jejuni from a dairy cow, retained its viability over eight weeks in pasteurized milk, while the most sensitive strain showed a > 5-log10 CFU/ml reduction in raw milk after one week. The majority (9/12) of the strains survived better in pasteurized milk than in raw milk, while the opposite was observed for two C. coli strains. The findings indicate that Campylobacter spp. can survive in milk for at least 14 days at 4oC and that survival is strain-dependent. To elucidate mechanisms underlying such adaptation, we characterized the role of capsule in Campylobacter survival in milk and other media, and in chick colonization. Three capsule-deficient mutants (kpsM::kanr), C. coli 6979M2, C. jejuni BS142M2 and C. jejuni SC1453M2 were constructed by site-specific mutagenesis. Compared to the wild type strains, the colonies of the mutants were smaller, more convex, and shinier on agar media; the mutants were more sensitive to desiccation and two (C. coli 6979M2 and C. jejuni BS142M2) grew slower in broth. These two mutants were also more sensitive to certain antibiotics than their wild type counterparts. The survival of the mutants in distilled water, raw and pasteurized milk was comparable to survival of their wild type counterparts, as was the ability of the bacteria to transform to nalidixic acid resistance via transformation. In chick colonization experiment, C. coli 6979 M2 showed > 3log10 reduced cell density in cecal contents than its parent strain. These results suggest that capsule is essential for C. coli in chick colonization and responsible for colony morphology on solid agar and can protect cells from desiccation, but its impact on growth rate, competence and antibiotic resistance is strain-dependent.
Temperature-dependent freeze-thaw tolerance and genetic characterization of stress response mechanisms of Listeria
(2009-11-08) Azizoglu, Reha Onur; Dr. Ilenys M. Pereze-Diaz, Committee Member; Dr. Sophia Kathariou, Committee Chair; Dr. Hosni Hassan, Committee Member; Dr. MaryAnne Drake, Committee Member
Listeria monocytogenes is a gram-positive foodborne pathogen that can cause severe illness (listeriosis) with high fatality rate. L. monocytogenes is ubiquitous in the environment and encounters a number of different stress conditions, including freeze-thaw stress. The growth temperature of the bacteria plays an important role in expression of several key virulence factors. However, our understanding of the impact of growth temperature on ListeriaÃ¢â‚¬â„¢s stress responses remains limited. In this study, we investigated the impact of growth temperature on the freeze-thaw tolerance of Listeria spp. Listeria cells grown at 37oC showed significantly higher tolerance against repeated freezing and thawing then cells grown at 4oC or 25oC (p<0.05). In order to address if this phenomenon is seen in other psychrotrophic foodborne pathogens, we investigated the impact of growth temperature on the freeze-thaw tolerance of Yersinia enterocolitica. However, impact of growth temperature on the cryotolerance of Y. enterocolitica was opposite of that observed with Listeria spp: Yersinia cells grown at 4oC were markedly more tolerant to the damaging effects of repeated freezing and thawing than 37oC-grown bacteria. To identify genes responsible for the observed temperature-dependent cryotolerance of L. monocytogenes we constructed mutant libraries of two L. monocytogenes strains, F2365 (serotype 4b) and 10403S (serotype 1/2a). The mutant libraries were constructed with a mariner-based transposition system and high efficiency of transposon insertion was achieved. However, screening directly for impaired cryotolerance following repeated freezing and thawing in a 96-well plate format did not allow consistent differentiation between 37oC-grown and 4oC-grown cultures. Therefore, our subsequent studies focused on screening for the loss of tolerance to the stresses expected to take place during repeated freeze-thaw treatments. The stresses included oxidative stresses, cold stress, and osmotic stress. One mutant in a putative oxidative stress gene was identified based on its susceptibility to paraquat and was found to have markedly impaired freeze-thaw tolerance. Thermoregulated control of cryotolerance resembles that of key virulence genes but is exhibited by both pathogenic and non-pathogenic species of Listeria. Growth temperature-dependent cryotolerance may represent an adaptation of ancestral Listeria lineages that preceded the emergence of non-pathogenic clades, and may involve thermoregulated attributes at the transcriptional or proteomic levels. The impact of growth temperature on cryotolerance of Listeria was unexpected, and underlying mechanisms remain to be identified.
Temperature-Dependent Phage Resistance in Listeria monocytogenes Epidemic Clone II Strains
(2008-12-02) Kim, Jae-Won; Dr. Sophia Kathariou, Committee Chair; Dr. Eric S. Miller, Committee Member; Dr. Todd R. Klaenhammer, Committee Member; Dr. Fred Breidt, Committee Member
Of at least 400 Listeria phages, only a few have been characterized as having wide host range among listeriae and no phages have been isolated from the environment of food processing plants, where contamination of highly processed, ready to eat foods is likely to occur. To understand the ecology of listeriaphage and Listeria monocytogenes in processing plant environments, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey-processing plants in the United States. Nine of twelve isolated phages showed wide host range and the majority of L. monocytogenes of the serotype 4b complex (serotypes 4b, 4d, 4e) could be readily infected by these wide host range phages, but many isolates of other serotypes (1/2a or 3a, and 1/2b or 3b), which represented the majority of L. monocytogenes isolated from the environmental samples, were resistant to infection. L. monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-99 and 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. We found that broad host range phages were unable to produce plaques on ECII strain lawns when the bacteria were grown at temperatures lower than 37Â°C (4, 10, 25, 30Â°C) , regardless of the temperature during infection and subsequent incubations. To identify genes responsible for this temperature-dependent resistance, use of a transposon led to successful isolation of a mutant (J46C), which was phage susceptible at both 25Â°C and 37Â°C. The ORF 2753, where the transposon was localized, and the adjacent ORF 2754 exhibited similarity with several restriction endonucleases and methylases, respectively, suggesting that they may constitute a restriction-modification system. Temperature-dependent phage resistance mechanisms may contribute to the ability of L. monocytogenes ECII to become established in the environment of processing plants, other environments characterized by relatively low temperatures, and foods. Further studies are needed to elucidate the molecular mechanism by which ECII-specific genes confer temperature-dependent resistance to phage, and to assess other possible roles of these genes in the ecology of the bacteria and during infection.