Enteric Diseases Program

• Identification and Serotyping
• Phage typing
• Molecular typing and characterization
• Surveillance
• Research

Acting Chief: Dr. Lai King Ng

The Enteric Diseases Program, formerly known as the NLEP (National Laboratory for Enteric Pathogens) is a  reference science capacity mandated since 1947 by Order-in-Council (PC857, 20 May, 1947). This program has been in continuous existence since that time.

The Program consists of five main components:

1. Identification and Serotyping
2. Phage typing
3. Molecular typing and characterization
4. Surveillance
5. Research

The Enteric Diseases Program provides diagnostic reference services, hazard identification, characterization of isolates, and surveillance for enteric pathogens. These include Salmonella, Shigella, Escherichia coli (including E. coli 0157:H7 causing “hamburger disease”), Campylobacter, Vibrio cholerae, Vibrio parahaemolyticus, Listeria monocytogenes, and Aeromonas.

Typing and sub-typing methods are necessary for characterization of disease-causing enteric bacteria. This information is used for outbreak identification, outbreak characterization and interventions to limit the impact of outbreaks on human populations. Surveillance of the types and subtypes of enteric bacteria circulating among humans, animals, food, and the environment also provides information vital for understanding how the human population is exposed to bacterial pathogens and how such exposures can be minimized or eliminated.

Many of the recent advances implemented within the Enteric Disease Program have been aimed at obtaining more accurate results more quickly, as well as using the most advanced web-based communication technologies and strategies to ensure the quickest response to public health threats caused by enteric bacteria.

The four components of the Enteric Disease Program work closely together, using methods that include biotyping, serotyping, phage typing, toxin typing, antibiotic testing and molecular typing.

This program as a whole also works closely with provincial Public Health Laboratories, other laboratories within the National Microbiology Laboratory, other federal laboratories working with food- and water-borne diseases, and with federal epidemiologists involved in surveillance and outbreak identification and control. Training in existing methodology is  provided by scientists within the Enteric Diseases Program, and  new methods for typing and subtyping of enteric bacteria are developed, tested, standardized, and shared. Research collaborations are developed within the Program and with external collaborators.

1. Identification and Serotyping.

Head: Helen Tabor

Identification of the bacterial genus and species is the first step in any bacteriological investigation. It is necessary to have this information to proceed with any other testing aimed at more precise characterization of bacterial isolates. The Identification and Serotyping group has the experience and expertise necessary to provide reference services in this area. This means that the methods used and results obtained by this section are accepted as the “gold standard” by other laboratories within the public health system.

This group is also responsible for serotyping enteric bacteria, a process that is critical for all subsequent investigations. Knowledge of the serotype of an enteric organism is often sufficient for outbreak identification, response, and control. Furthermore, the tests used for further characterization of these bacteria depend on serotype, and most other typing and subtyping information obtained on bacteria of interest is organized according to serotype. The Identification and Serotyping laboratory is one of only a very few in the world with the experience, expertise, and reference bacteria collections to prepare and perform rigorous quality control testing on rabbit antiserum for the serotyping of Salmonella, Shigella, E. coli, and Listeria monocytogenes. Some of this antiserum is subsequently provided to laboratory partners at the national and international level through Material Transfer Agreements. All methods used are those recommended by the Institut Pasteur and the WHO Collaborating Centres. The personnel and expertise available within the Identification and Serotyping laboratory have been used by the World Health Organization for training laboratory scientists in other countries in bacterial identification and serotyping. This laboratory has been heavily involved the development of serotyping laboratories, reagents, culture collections, and quality assurance programs, especially in Latin America. It has also developed and delivered quality assurance programs to the Canadian Public Health Laboratory Network.

2. Phagetyping

Head: Rafiq Ahmed

Phage typing is a very rapid, highly discriminatory subtyping method that can quickly provide information about bacterial strains within serotypes. It has proved to be an economical, robust and informative method for the identification and control of outbreaks in situations where serotyping alone does not provide sufficient information. Phage typing has also provided highly informative results for the study of bacterial populations. The Phage Typing laboratory of the Enteric Disease Program engages in laboratory testing, phage production, applied research and surveillance activities. All of these activities provide information important for the identification of emerging public health issues, for priority setting, and for the establishment of departmental research projects. Several new phage typing methods have been developed recently to address gaps in our ability to subtype important human enteric pathogens. These include
E. coli O157:H7, B. cereus, Salmonella Heidelberg, Salmonella Oranienburg, Salmonella Newport, Shigella sonnei, and Shigella boydii.

Phage reagents and training in phage typing for E.coli O157:H7 and  Methicillin Resistant Staphylococcus aureus (MRSA) have been provided to external clients and representatives from national and international health centers.

3.  Molecular typing and characterization

Dr. Gehua Wang

Another service provided to public health laboratories is to detect or characterize toxins produced by enteric pathogens, e.g. shiga-toxins, cholera toxins. Atypical isolates may require the detection of virulence factors or unique genes to confirm their identity or pathogenicity.

4. Surveillance

Walter Demzuk

National Enteric Surveillance Program (NESP)

The National Enteric Surveillance Program (NESP) is a surveillance system for monitoring short term fluctuations in the numbers of human enteric pathogen isolates in Canada to quickly identify outbreaks of disease. The audience for this information includes federal and provincial individuals directly involved in the identification, prevention and control of enteric disease in Canada. The basic data sets are provided to the NML by each of the provincial Public Health Laboratories and are collated and analyzed at the NML and in the CIDPC in Guelph.  These data change from week to week to incorporate updates and corrections. Annual NESP data are useful to determine background levels and baselines necessary to put the weekly isolation totals into context.

Enteric Surveillance Annual Summaries

The National Enteric Summary reports are made more accurate and broadly-based than NESP data collections by including and comparing data from several sources. These sources include the National Notifiable Disease data supplied by Centre for Infectious Disease Prevention and Control, NESP, and Canadian Integrated Program for Antimicrobial Resistance Surveillance, our own reference services, and data that the LFZ shares on Salmonella isolates from non-human sources.  The National Enteric Summary is a more accurate and in-depth study of the levels of enteric pathogen isolations in Canada. This report provides context and interpretation of the levels, possible sources, and distribution of enteric pathogens in Canada, and is intended for domestic and international public health agencies from a public health management perspective .  It has initiated enhanced surveillance programs such as the S. Newport project as part of the Canadian Integrated Program for Antimicrobial Resistance (CIPARS) Surveillance.  It has also identified trends such as the increase in S. Heidelberg as a cause of gastrointestinal illness in Canada and, although non-human data is limited, has identified poultry as a reservoir for this organism.  Surveys have been initiated such as the chicken nugget study that resulted in public advisories and information campaigns aimed at the proper handling and safe cooking guidelines of these products.

Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS)

In 2002, the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) was initiated to monitor trends in antimicrobial use and antimicrobial resistance in selected bacterial organisms from human, animal and food sources across Canada. CIPARS is a national antimicrobial resistance and antimicrobial use surveillance program dedicated to the collection, integration, analysis and communication of timely, ongoing and representative information derived from animals, foods, humans and the environment. The program is based on several representative and methodologically unified surveillance components which can be linked to examine  the relationship between antimicrobials used in food animals and humans and the associated health impacts. This knowledge will support (i) the creation of science-based policies to control antibiotic use in hospital, community and agricultural settings and thus prolong the effectiveness of these drugs and (ii) identification of appropriate measures to contain the emergence and spread of resistant bacteria between animals, food and  people. The ultimate objective is to mitigate the impact of antimicrobial resistance on the Canadians.

The more detailed objectives of this program are to examine the magnitude and trends in antimicrobial resistance in human enteric clinical isolates across Canada and identify emerging issues. CIPARS surveillance of antimicrobial resistance in human isolates of Salmonella, a collaboration between the Canadian Public Health Laboratory Network and the Public Health Agency of Canada, began in January 2003. Provincial public health laboratories forward a representative sample of Salmonella isolates with accompanying data  to the National Microbiology Laboratory for phagetyping and susceptibility testing.

PulseNet Canada
 PulseNet Canada is a network of laboratories sharing laboratory subtyping results in real-time using internet and web-based information transfer technologies. The creation of national continuous, comprehensive databases of bacterial types using standardized methods and a standardized nomenclature is co-ordinated and monitored by the PulseNet group within the National Microbiology Laboratory.

The sudden appearance of a few isolates of a new type or an increase in incidence of a previously identified type identifies a cluster of cases that could be an outbreak, precipitating a laboratory and epidemiological investigation. Molecular typing (PFGE) and other typing and subtyping data can then be used to refine the case definitions to confirm vehicles of infection and identify routes of infection. Results from typing and subtyping methods are also valuable for trace-back investigations.

Pulsed Field Gel Electrophoresis (PFGE) has been used in many clinical, food and reference laboratories in Canada to genotype bacterial pathogens strains since the early 1990's.  The desire to compare results between laboratories to detect outbreaks of multi-geographical regions brought a group of laboratory managers across the country to the National Microbiology Laboratory in 1998 to standardize PFGE protocols.  By consensus, the E. coli O157:H7 standard method from the Center for Disease Control and Prevention was adopted.  In 2000, PulseNet North was born which was renamed as PulseNet Canada in 2003.  The current PFGE method used by PulseNet Canada laboratories has been harmonized with those used by CDC. To ensure consistency of inter-laboratory results, all laboratories within the network participate in the quality assurance program.

For preparedness and response to natural and intentional outbreaks of infectious disease, improved methods of collection and dissemination of information across different jurisdictions are required.  As a result, NML initiated a virtual framework the Canadian Laboratory Surveillance Network (CLSN) for molecular epidemiology of infectious disease agents that may have national and international impact.  In consultation with the provincial and federal laboratories, PulseNet Canada is placed under the umbrella within this framework.  PulseNet Canada memberships include the Microbiology Laboratory (NML) and the Laboratory of Foodborne Zoonoses (LFZ) of the Public Health Agency of Canada (PHAC), the Bureau of Microbial Hazards, Food Directorate, Health Canada, and the provincial laboratories of the CPHLN.

5. Research

Dr. Clifford Clark and Dr. Gehua Wang

Most research performed on enteric organisms falls into four categories: 1) development of improved detection methods, 2) assessment and development of better typing methods, 3) special laboratory surveillance projects and analysis of outbreaks and outbreak strains, and 4) investigations into the distribution of virulence genes within populations of these organisms and mechanisms by which these genes are moved through the population.

The development of improved detection methods includes the creation of new polymerase chain reaction (PCR) tests for detecting signature virulence genes, multiplex PCR techniques for rapid identification of E. coli pathotypes, and real-time PCR methods for use in mobile laboratories. Collaborative work is aimed at the development of molecular serotyping methods that can be used in any laboratory. Novel rapid methods under investigation include the Luminex bead technology for the identification of sets of up to100 genes and DNA microarrays for the identification of much larger gene sets. A current interest is the use of real-time multiplex PCR to characterize the diversity and estimate the relative numbers of each enteric bacterial pathogen in stools and other specimens from human disease. The intent is to develop better algorithms for identification of the causative agent(s) of disease.

The utility and predictive power for epidemiological relationships of several different typing and subtyping methods has been evaluated for isolates involved in the Walkerton Campylobacter waterborne outbreak. Similar investigations are planned or underway for other enteric pathogens. This work will allow more accurate outbreak identification and facilitate the epidemiological characterization of outbreaks and trace-back investigations. Typing and subtyping methods are also being used to characterize the bacterial diversity of E. coli associated with diarrheal disease in young children in Manitoba.Other research is focused on the biology of enteric pathogens. Current projects include the identification of novel virulence genes in enteric organisms, the determination of mechanisms by which virulence genes are moved within populations of enteric bacteria, and the association of specific lineages of enteric bacteria with disease in humans. These investigations will answer questions about what genes are necessary for each pathogen to cause disease and what genetic mechanisms are operating to create new pathogens.