# Presentation: 094218

## Using the Tools of Molecular Biology to Aid in Foodborne Disease Investigations

- Leslie A. Wolf, PhD
- North Carolina State Laboratory of Public Health
- Raleigh, NC
- May 14, 2002

## Overview of Presentation

- Introduce key molecular biology terms
- Discuss a variety molecular subtyping techniques
- Explain PulseNet
- Highlight a number of foodborne disease outbreak investigations that used molecular subtyping for epidemiologic purposes

## General Molecular Biology Terms

- Genome
- DNA
- RNA
- Gene
- PCR
- Primers
- Restriction Enzyme
- Fingerprinting
- Mutation

## Genome

- The entire nucleic acid molecule of an organism that encodes enzymes, proteins, and other structural components
- For most bacteria, a single circular molecule containing DNA

## DNA

- Deoxyribonucleic acid
- Sugar-phosphate backbone and nitrogenous bases form the helical double-stranded molecule
- Adenosine pairs with Thymidine
- Guanosine pairs with Cytosine

## RNA

- Ribonucleic acid
- Contains Uracil instead of Thymidine
- Messenger RNA (mRNA) encodes proteins
- Transfer RNA (tRNA) chooses correct amino acid to build proteins
- Ribosomal RNA (rRNA) provides docking structure for protein assembly

## mRNA/tRNA

## Ribosomal RNA

## Gene

- A portion of the genome that encodes a specific product, such as a protein or enzyme or other macromolecule
- Example:  the gene *stx*1 encodes a shiga toxin from *E. coli* O157:H7

## Primers

- Short pieces of DNA that bind to sequences of denatured DNA
- Sometimes designed to be highly specific to target a certain gene
- Sometimes designed to be degenerate to increase chances for binding

## PCR

- Polymerase Chain Reaction
- Method developed by K. Mullis as a way to copy DNA *in vitro*
- One copy of a gene can be amplified 106 times so that it becomes “visible” by gel electrophoresis or by fluorescent assays

## Restriction Enzyme

- Enzymes produced naturally by many strains of bacteria as a defense mechanism
- Enzymes cut DNA at specific combinations of A, G, C and T.
- Named systematically: EcoR1 is the first enzyme isolated from *Escherichia coli*

## Fingerprinting

- A term used to describe any method that provides additional information at the molecular level to distinguish among bacterial strains

## Mutation

- A change in the nucleic acid sequence that may or may not be readily observed
- (lethal 	      neutral)
- Insertion of A, T, G or C
- Deletion of A, T, G or C
- Substitution of bases (point mutations)
- Inversions

## Pulsed-Field Gel Electrophoresis

- PFGE is gold standard for bacterial subtyping
- Looks at whole genome of bacterial pathogens using rare cutting restriction enzymes
- Fragments are 30 kb to several hundred kb in size
- Labor-intensive, but readily standardized

## PFGE

**Bacterial Chromosome**

**With Rare Restriction Sites**

**DNA Fingerprint**

**Enzyme Digest**

**Electrophoresis**

## Restriction Fragment Length Polymorphism

- RFLP is a method of detecting changes in restriction sites at specific genetic loci
- Fragments are generally less than 1 kb
- Useful only for genes that are variable

## Ribotyping

- Automated system available from Dupont-Qualicon
**(www.qualicon.com)**
- Variation of RFLP, using primers to rRNA gene targets
- Chemiluminscent detection of banding patterns due to probe binding sites

## Randomly Amplified Polymorphic DNA

- RAPD uses short random sequence primers, 9-10 bases long
- Amplifies chromosomal DNA at low annealing temperatures
- Number and location of random primer sites vary for different strains

## Repetitive Extragenic Palindromic Element PCR

- PCR based amplification of REP or ERIC sequences
- Short amplicons, 38 or 126 bp in length
- Highly conserved and widely dispersed in enteric bacterial genomes (J. Versalovic
*et. al.*, 1991, *Nucleic Acids Research* and www.bacbarcodes.com)

## Rep-PCR

## Rep-PCR

## Rep-PCR

## Rep-PCR

## Rep-PCR

## Next Generation Subtyping Methods

- DNA Sequencing
- MLST
- VNTR
- MLVA
- PATS
- Microarray

## Multilocus Sequence Typing

- PCR amplification of several housekeeping genes
- PCR amplification of one or more variable genes
- DNA sequencing of amplification products

## PulseNet

- The National Molecular Subtyping Network for Foodborne Disease Surveillance

## PulseNet

- Initiated in 1998 in response to a large outbreak of *E. coli* 0157:H7 in western US
- Standardized PFGE procedures allow participating laboratories to rapidly compare results electronically
- Certification and proficiency testing are required of all participating laboratories

## PulseNet

- Participants include CDC, state public health laboratories, NYC, Houston, and LA city laboratories, FDA and USDA laboratories
- Seven area laboratories provide additional capacity (WA, UT, TX, MN, MI, VA, MA)
- National databases for *E. coli* O157:H7, *Salmonella,* *Listeria monocytogenes* and *Shigella sonnei* are managed by CDC

## PulseNet

- Goals of national surveillance system are to identify clusters of foodborne disease and to assist in epidemiologic investigations
- Ideally this will limit foodborne outbreaks by identifying sources of contamination and by raising awareness of food safety issues

## Escherichia coli O157:H7

- SEM of *Escherichia coli* ©Scott Kachlany, author.  Licensed for
- Use, ASM MicrobeLibrary

## E. coli O157:H7

- Gram-negative rod, producing shiga toxins 1 or 2, low infectious dose
- Causes nausea, abdominal cramping and frequently, bloody diarrhea
- Elderly and young children most at risk of severe illness; HUS is a serious complication in young children
- A number of food vehicles identified during outbreaks, including hamburger, lettuce, and unpasteurized apple juice

## Listeria monocytogenes

- SEM of *Listeria monocytogenes* ©Amy C. Lee Wong, author.  Licensed for
- Use, ASM MicrobeLibrary

## Listeria monocytogenes

- Gram positive rod, intracellular pathogen
- Causes sepsis or meningitis in immuno-compromised hosts; causes a mild, flu-like illness in pregnant women; febrile gastroenteritis in immunocompetent hosts
- Causes spontaneous abortions, premature births, and newborns with bacteremia, leading to fetal deaths
- Soft cheeses, unpasteurized milk products, and ready to eat deli meats are common vehicles of transmission

## Salmonella enterica

- Gram negative rod, 2500 serotypes
- Sudden onset of illness, causing fever, nausea, abdominal pain and diarrhea for 4-7 days
- Second most common cause of bacterial gastroenteritis in US, usually in infants and young adults
- A number of food vehicles have been implicated in outbreaks, including toasted oat cereal, cantaloupe, mangoes, and eggs

## Campylobacter jejuni

- Microaerophilic gram negative,”seagull” shaped bacterium
- Prodrome of 1-2 days with fever, headache, muscle pain and malaise with acute onset of diarrhea, cramping, abdominal pain, fever
- High percentage of raw poultry contaminated with *C. jejuni; *raw milk may be contaminated
- Most common cause of bacterial gastroenteritis in US, usually infants and young adults; mostly sporadic cases, few outbreaks

## Shigella sonnei

- Gram negative rod, low infectious dose
- Most common in daycare settings, no animal reservoir
- Causes acute onset of watery or bloody diarrhea, nausea, abdominal pain, fever, malaise lasting 4-7 days
- Some foodborne outbreaks have occurred in recent years, including parsley and lettuce

## Calicivirus

## Calicivirus/Norwalk-like Viruses

- Non-cultivable RNA viruses, only detected by electron microscopy and reverse-transcriptase PCR
- Many foodborne outbreaks, particularly in oysters contaminated with raw sewage, and via restaurants and catering establishments
- Nausea, vomiting, abdominal cramps and diarrhea for 24-48 hours; headache and low grade fever may also occur

## MMWR 50(RR09): 1-18, 2001

## E. coli O157:H7 outbreak, Robeson County, NC 2001

- November 28th, physician reports to RCHD that several children had symptoms of bloody diarrhea
- Culture confirmed 11 children had O157:H7
- PFGE analysis of first several isolates showed indistinguishable patterns with two enzymes
- Between mid-November and late December, 203 suspect cases of *E. coli* O157:H7 infection were identified

## E. coli O157:H7 outbreak

- Two events held at local schools:  food tasting for Native American Cultural Heritage month and tasting of homemade butter
- After interviewing confirmed cases and controls, three suspect foods emerged:  hamburger, string beans and butter

## E. coli O157:H7 outbreak

- After controlling for exposure to hamburger and string beans, a highly significant association between illness and eating butter made with unpasteurized milk remained
- Manure from milk cows tested positive for shiga toxin by EIA

## E. coli O157:H7 PFGE Patterns

- 100

- 90

- 80

- 70

- 60

- 50

- A

- A1

- A2

- A3

- B

- C

- XbaI patterns

- AvrII patterns

- A

- A

## Recommendations

- Educate school age children about proper handwashing techniques
- Examine school exclusion policies for illness in different age groups
- Discourage production and distribution of home-made food made from raw milk products

## L. monocytogenes outbreak, Forsyth County,NC 2000

- 12 cases in hispanic population between October 2000 and January 2001
- 11 were females, 10 were pregnant
- Infections with *L. monocytogenes* led to 5 stillbirths, 3 premature deliveries and 2 infected newborns
- Illness associated with eating cheese purchased from door-to-door vendors

## L. monocytogenes outbreak

- Investigation of these cases revealed that Mexican-style soft cheese was made in private homes from raw milk
- Home-made cheese sold door-to-door or in parking lots
- Found unlabeled home-made cheese in local Latino grocery stores

## L. monocytogenes PFGE Patterns

- AscI digest

- ApaI digest

- Serotype 4b

**Ribotyping patterns were indistinguishable:  DUP1042**

## Recommendations

- Limit sale of raw milk products to regulated processors
- Educate hispanic communities about the dangers of eating unpasteurized cheeses while pregnant
- Make listeriosis a reportable illness in North Carolina
*MMWR* 50(26): 560-562, July 6, 2001

## North Carolina Salmonella Enteritidis Case Numbers by Month, 2001

**Month of Collection**

## Age Distribution of S. Enteritidis cases in North Carolina, 2001

**Age, in years**

## Number of Isolates with Common

*S. *Enteritidis PFGE Patterns, 2001

## Common S. Enteritidis PFGE Patterns, North Carolina 2001

- 100

- 95

- 90

- -220

- -225

- -230

- -235

- -240

- -245

- -250

- -255

- -260

- -265

- -270

- -275

- -280

- -285

- -290

- -295

- -300

**C**

**B**

**D**

**J1**

## Conclusions

- PFGE subtyping of *S*. Enteritidis isolates during an outbreak is useful for separating outbreak from non-outbreak cases
- Additional subtyping of *S*. Enteritidis, such as phage typing, may be required
- Safe handling of eggs, particularly at the point of consumption, is necessary to prevent infection with *S*. Enteritidis

## Norwalk-like virus outbreak, Texas 1998

- March 1998, 23 students from local university treated in ER for acute gastroenteritis
- Investigation showed that illness was associated with eating at the campus cafeteria or deli bar during lunch/dinner
- Food handler who prepared deli ham and sandwiches wore gloves, but took care of infant with watery diarrhea two days before preparing food

## NLV outbreak, Texas

- 50% of stools submitted by students demonstrated evidence of NLV by reverse transcriptase PCR
- The only food that tested positive for NLV by RT-PCR was deli ham
- Sequence analysis of NLV from ill students, deli ham, and the infant of food handler had identical sequences in the capsid gene
*Journal of Infectious Diseases*, 2000, 181:1467-1470

## Recommendations

- Closed deli bar once became suspected as source of outbreak
- Consider paid leave for food handlers with gastroenteritis
- Further studies needed to determine duration of shedding infectious NLV
- Educate food handlers regarding personal hygiene when caring for family members (especially diapered infants) with gastroenteritis

## Questions?