Division of Foodborne, Waterborne, and Environmental Diseases

 

1. DFWED Pathogenesis, Evolution, and Comparative Genomics of Salmonella serotypes

P. I. Fields

Salmonella is one of the most common foodborne pathogens in the US. It typically causes gastrointestinal illness, but specific serotypes are associated with invasive disease. Additionally, some serotypes are associated with specific animal hosts. Our current knowledge of the genetics and pathogenesis of Salmonella is restricted to just a few common serotypes. However, "Salmonella" consists of two species, seven subspecies, and more than 2500 different serotypes; and, some serotypes have been shown to differ by more than 10% in gene content. Our research is focused in two areas. We are studying the genetic basis for serotype variability, and are applying this knowledge to the development of molecular methods for the identification of serotype. And, we are characterizing specific Salmonella serotypes with respect to genome content and/or genome sequence in order to better understand the evolution of Salmonella, and the pathogenic and epidemiologic differences between serotypes. A better understanding of the pathogenesis and diversity of Salmonella serotypes can be applied the development of improved diagnostic and subtyping methods, as well as to prevention and control strategies.

 

2. DFWED Development of Novel Molecular Methods for Subtyping Bacterial Foodborne Pathogens

 

E. Hyytia-Trees, E. M. Ribot

 

An estimated 48 million illnesses are caused by foodborne pathogens in the United States each year resulting in approximately 128,000 hospitalizations and 3,000 deaths. An estimated 3.6 million illnesses are attributed to foodborne bacterial pathogens such as Shiga-toxin producing Escherichia coli (STEC), Listeria, and Salmonella spp, among others. We are interested in developing and applying novel DNA-based molecular subtyping methods, especially next generation sequencing, as part of our efforts to improve the way we characterize and study these human pathogens and measure the impact they have on public health. Our primary objective is to develop cutting-edge subtyping tools for use by PulseNet, CDC’s National Molecular Subtyping Network for Foodborne Disease Surveillance, that are based on comparative sequencing and/or analysis of multiple loci such as housekeeping and virulence genes, tandem repeats, and single nucleotide polymorphisms. We are also interested in developing culture-independent approaches (focus on metagenomics) for the identification and characterization of foodborne pathogens directly from clinical specimens to further enhance PulseNet’s laboratory-based surveillance activities.

 

References

 

Lanier, W.A., Hall, J.M., Herlihy, R.K., Rolfs, R.T., Wagner, J.M., Smith, L.H., and Hyytia-Trees, E.K. (2011) Outbreak of Shiga-toxigenic Escherichia coli O157:H7 infections associated with rodeo attendance, Utah and Idaho, 2009. Foodborne Path. Dis. 8, 1-3.

 

Hyytia-Trees, E., Lafon, P., Vauterin, P., and Ribot, E.M. Multilaboratory Validation Study of Standardized Multiple-Locus Variable-Number Tandem Repeat Analysis Protocol for Shiga Toxin–Producing Escherichia coli O157: A Novel Approach to Normalize Fragment Size Data Between Capillary Electrophoresis Platforms. 2010. Foodborne Pathogens and Disease. 7:129-136

 

Zhang, W. et al. 2007. Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms. Genome Res. 16: 757-767.

 

Hyytia-Trees, E., Smole, S.C., Fields, P.A., Swaminathan, B., and Ribot, E.M. 2006. Second Generation Subtyping: A Proposed PulseNet Protocol for Multiple-Locus Variable-Number Tandem Repeat Analysis of Shiga Toxin–Producing Escherichia coli O157 (STEC O157). Foodborne Pathogens and Disease. 3:118-131.


Ribot, E.M., Fair, M.A., Gautom, R, Cameron, D.N., Hunter, S.B., Swaminathan, B., Barrett, T.J. 2006. Standardization of Pulsed-Field Gel Electrophoresis Protocols for the Subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathogens and Disease. 3:59-67.

 

3. DFWED Molecular Epidemiology of Waterborne and Foodborne Protozoa

L. Xiao

Research in the laboratory focuses on the development of molecular tools at species, genotype and subtype levels, and usage of these tools in investigating the transmission of Cryptosporidium, Giardia, Cyclospora, and microsporidia in humans and animals, and in detecting and tracking these organisms in environmental samples. Specific research areas include (1) molecular taxonomy of Cryptosporidium, Giardia, Cyclospora, and microsporidia in the context of public health, (2) assessment of the public health importance of zoonotic parasites, (3) determination of risk factors, infection sources, and pathogen spread in outbreak and endemic settings, (4) population genetics and transmission dynamics of enteric parasites, and (5) assessment of source and human infection potential of oocysts/cysts/spores in water, food, and environment.

References

Feng, Y., Yang, W., Ryan, U., Zhang, L., Kvac, M., Koudela, B., Modry, D., Li, N., Fayer, R., Xiao, L., 2011, Development of a multilocus sequence tool for typing Cryptosporidium muris and Cryptosporidium andersoni. J Clin Microbiol 49, 34-41.

Feng, Y., Li, N., Dearen, T., Lobo, M.L., Matos, O., Cama, V., Xiao, L., 2011, Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi. Appl Environ Microbiol 77, 4822-4828.

Feng, Y., Zhao, X., Chen, J., Jin, W., Zhou, X., Li, N., Wang, L., Xiao, L., 2011, Occurrence, source, and human infection potential of Cryptosporidium and Giardia spp. in source and tap water in Shanghai, China. Appl Environ Microbiol 77, 3609-3616.

Wang, R., Wang, H., Sun, Y., Zhang, L., Jian, F., Qi, M., Ning, C., Xiao, L., 2011, Characteristics of Cryptosporidium transmission in pre-weaned dairy cattle in Henan, China. J Clin Microbiol 49:1077-1082.

Feng, Y., Xiao, L., 2011, Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 24, 110-140.

 

4.  DFWED Application of Bioinformatics Tools to Improve Subtyping of Bacterial Foodborne Pathogens

M. M. Freeman

Foodborne diseases are a significant burden on the health care system, with around 76 million Americans becoming ill annually.  The main bacterial culprits are shiga-toxin producing Escherichia coli (STEC), Salmonella, Shigella, Campylobacter and Listeria monocytogenes.  These organisms are tracked by PulseNet, CDC’s National Molecular Subtyping Network for Foodborne Disease Surveillance.  PulseNet identifies clusters of similar strains based on patterns generated by PFGE (pulsed-field gel electrophoresis).  While PFGE remains the “gold-standard” for subtyping bacterial foodborne pathogens, new molecular techniques continue to be explored by our group.  Next generation methods for molecular characterization and subtyping, ideally, would take less time, cost less and be more discriminatory and epidemiologically relevant than PFGE.  Additionally, being able to link data generated by new methods to existing data is necessary to validate the new method.  The PulseNet database of PFGE patterns spans over 14 years and 8 different organisms and contains corresponding isolate and demographic data that is amendable to data mining projects.   Hypothesis-driven searches may uncover previously unknown trends in bacterial diseases and outbreaks that could explain past events or help prevent future outbreaks.  Bioinformatics tools can point to new ways to analyze these data and other information associated with over 300,000 PFGE fingerprints currently contained in the PulseNet national database.  Finally, we aim to apply informatics to help interpret genomic data in the context of bacterial foodborne outbreaks.  Powerful analytical tools should shed light on new methods or uncover novel approaches subtype strains. 

References

Zhang, W. et al. 2007. Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms. Genome Res. 16: 757-767.

Hyytia-Trees, E., Smole, S.C., Fields, P.A., Swaminathan, B., and Ribot, E.M. 2006.  Second Generation Subtyping: A Proposed PulseNet Protocol for Multiple-Locus Variable-Number Tandem Repeat Analysis of Shiga Toxin–Producing Escherichia coli O157 (STEC O157). Foodborne Pathogens and Disease. 3:118-131.

Ribot, E.M., Fair, M.A., Gautom, R, Cameron, D.N., Hunter, S.B., Swaminathan, B., Barrett, T.J. 2006. Standardization of Pulsed-Field Gel Electrophoresis Protocols for the Subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathogens and Disease. 3:59-67.

 

5. DFWED Environmental Microbiology Investigations of Waterborne and Environmental Diseases

 

V.R. Hill

 

This laboratory serves as a core CDC resource for development and implementation of environmental microbiology techniques with two general focus areas: 1) recovery and detection of pathogens and microbial indicators of fecal contamination in environmental samples and 2) survival and inactivation of microbes in environmental systems (e.g., drinking water). The Environmental Microbiology Laboratory works with Global and Domestic Water, Sanitation, and Hygiene (WASH) programs, as well as subject-matter expert programs focusing on specific pathogens and diseases. The laboratory has capacity and expertise in applying environmental microbiology techniques to the study of bacteria, viruses, and parasites in environmental samples. Techniques employed include sample collection (e.g., microfiltration, ultrafiltration), sample processing (e.g., centrifugation, immunomagnetic separation), bacterial culture (agar and broth), bacteriophage plaque assays, tissue culture for virus quantification and parasite infectivity analysis, fluorescence microscopy, nucleic acid extraction and molecular amplification of DNA and RNA (e.g., qPCR, RT-qPCR, isothermal amplification), and analysis of molecular products (e.g., pyrosequencing, Luminex liquid phase microarrays). Disinfection research includes work on recreational water applications (e.g., hyperchlorination of swimming pools to inactivate Cryptosporidium) and drinking water applications (free chlorine and monochloramine inactivation of enteric viruses).

 

References

 

Yoder JS, Straif-Bourgeois S, Roy SL, Moore TA, Visvesvara GS, Ratard RC, Hill VR, Wilson JD, Linscott AJ, Crager R, Kozak NA, Sriram R, Jothikumar N, Mull B, Kahler AM, Schneeberger C, da Silva AJ, Beach MJ. (2012) Primary Amebic Meningoencephalitis Deaths Associated with Sinus Irrigation Using Contaminated Tap Water. Clin. Inf. Dis., 54:805-809.

 

Prithiviraj J, Hill VR, Jothikumar N. (2012) Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay. Biochem. Biophys. Res. Comm., 420 :738-742.

 

Hill VR, Cohen N, Kahler AM, Jones JL, Bopp CA, Marano N, Tarr CL, Garrett NM, Boncy J, Henry A, Gómez GA, Wellman M, Curtis M, Freeman MM, Turnsek M, Benner Jr RA, Dahourou G, Espey D, DePaola A, Tappero JW, Handzel T, and Tauxe RV. (2011) Toxigenic Vibrio cholerae O1 in water and seafood, Haiti. Emerg. Infect. Dis., 17:2147-2150.

 

Hill VR, Mull B, Jothikumar N, Ferdinand K, and Vinje J. (2010) Detection of GI and GII Noroviruses in Ground Water Using Ultrafiltration and TaqMan Real-time RT-PCR. Food Environ. Virol., 2:218-224.

 

Kahler AM, Cromeans TL, Roberts JM and Hill VR. (2010) Effects of source water quality on chlorine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus. Appl. Environ. Microbiol., 76:5159-5164.

 

Smith CM and Hill VR. (2009) Dead-end hollow fiber ultrafiltration for recovery of diverse microbes in water. Appl. Environ. Microbiol., 75(16):5284-5289.

 

Shields JM, Hill VR, Arrowood MJ, and Beach MJ. (2008) Inactivation of Cryptosporidium parvum under chlorinated recreational water conditions. J. Water Health, 6(4):513-520


6. DFWED Comparative Genomics of Botulinum Toxin Producing Bacteria

B. H. Raphael

Foodborne botulism is a rare and potentially fatal disease resulting from the ingestion of a paralytic neurotoxin. Botulism is also reported in infants and in individuals with wounds where the organism colonizes and produces toxin the gut or other tissues, respectively. Because of the extreme potency of botulinum neurotoxins, they are considered a potential bioterrorism agent. The National Botulism Laboratory Preparedness Team (NBLPT) is engaged in applied research activities involving: 1) the detection of neurotoxin in various matrices, 2) the detection of botulinum toxin producing clostridia using molecular methods, and 3) novel methods for the molecular subtyping of botulinum toxin producing clostridia. Opportunities exist for development of DNA microarrays as a strain subtyping method and as an approach to provide insights into the phylogenetic relationships among botulinum toxin producing clostridia.

References

Raphael BH, Luquez C, McCroskey LM, Joseph LA, Jacobson MJ, Johnson EA, Maslanka SE, Andreadis JD. 2008. Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters. Appl Environ Microbiol. 74(14):4390-7.

Raphael, BH, Choudoir MJ, Luquez CL, Fernandez R, Maslanka S. 2010. Sequence diversity of genes encoding botulinum neurotoxin type F. Appl Environ Microbiol. 76(14):4805-12.


7. DFWED Investigation of antifungal resistance in yeasts and molds

S. R. Lockhart

Infections due to the yeast Candida are the third most prevalent nosocomial bloodstream infections and cause considerable morbidity and mortality.  Mold infections, especially Aspergillus spp., are a leading cause of morbidity and mortality among transplant patients. The antifungal armamentatrium to fight these infections is limited and the organisms are capable ofdeveloping new mechanisms of resistance.  The Antifungal Research Unit of the Mycotic Diseases Branch performs surveillance for these fungal infections and specializes in antifungal susceptibility testing of yeasts and molds. We are interested in surveillance for antifungal drug resistant yeasts and molds, especially Candida, Cryptococcus and Aspergillus, as well as characterizing the molecular mechanisms responsible for this resistance.  Our group has recently reported on the susceptibility of Candida and Aspergillus isolates from US transplant patients, the presence of specific mutations in Aspergillus fumigatus isolates that render them resistant to itraconazole and the molecular mechanisms of echinocandin resistance in isolates of Candida glabrata. Current research includes US candidemia surveillance, surveillance for Cryptococcus gattii in the Pacific northwest and the establishment of species-specific antifungal breakpoints for uncommon Candida species. Opportunities are available for a fellow to explore mechanisms of antifungal resistance, especially in Candida and Aspergillus.  We are also interested in exploring the molecular mechanisms and possible genome rearrangements responsible for fluconazole and echinocandin co-resistance in Candida glabrata.

References

Lockhart S.R., J.P. Frade, K.A. Etienne, M.A. Pfaller, D.J. Diekema and S.A. Balajee. (2011) Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance is primarily due to the TR/L98H mutation in cyp51A. Antimicrobial Agents and Chemotherapy, 55:4465-4468.

Lockhart, S.R., A.J. Zimbeck, C.B. Bolden, J.W. Baddley, K.A. Marr, D.R. Andes, T.J. Walsh, C.A. Kauffman, D.P. Kontoyiannis, J.I. Ito, P.G. Pappas and T.C. Chiller. (2011) In vitro susceptibility of Aspergillus isolates to echinocandins from patients enrolled in the Transplant-Associated Infection Surveillance Network. Antimicrobial Agents and Chemotherapy, 55:3944-3946.

Lockhart, S.R., N. Iqbal, D. Wagner, P.G. Pappas, D.R. Andes, C.A. Kauffman, L.M. Brumble, S. Hadley, R. Walker, J.I. Ito, J.W. Baddley, T. Chiller, and B.J. Park. (2011) Comparison of in vitro susceptibility characteristics of Candida species from cases of invasive candidiasis in solid-organ and stem cell transplant recipients: Transplant-Associated Infections Surveillance Network (TRANSNET) 2001-2006. Journal of Clinical Microbiology, 49:2404-2410.

Zimbeck, A., N. Iqbal, A.M. Ahlquist, M.M. Farley, L.H. Harrison, T. Chiller, and S.R. Lockhart (2010). FKS mutations and elevated echinocandin MIC values among Candida glabrata isolates from US active surveillance. Antimicrobial Agents and Chemotherapy. 54:5042-5047.

 

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