TYPING METHODS
WHAT IS TYPING AND WHAT ARE TYPING METHODS? Pathogenic bacteria replicate and persevere in ecological niches called reservoirs Reservoirs may be humans, including (fellow) patients and healthcare personnel, animals, plants, water, food and various niches in the environment
Transmission of bacteria from any of these sources may generate clusters of colonization or infection among humans Such clusters are recognized mostly as outbreaks of infectious diseases When these outbreaks are not controlled, major epidemics (due to unrestricted further transmission) may arise Bacterial epidemiological typing generates isolate-specific genotypic or phenotypic characters that can be used to elucidate the sources and routes of spread of bacteria
The scope of typing studies Clinical : (dissemination of infections from patients, animals or other sources to non-colonized and uninfected individuals) Environmental: (the presence or spread of organisms in inanimate surroundings) or even industrial (identification of organisms that are either valuable or a menace to bio-industry)
Different levels of Typing Locally, at a hospital or other primary laboratory Regionally or nationally, in a reference laboratory to bear upon wider issues of public health and surveillance Internationally through collaborative networks, to define or survey the worldwide dissemination of major bacterial clones
TYPING TERMS Bacterial epidemiology: The study of the dissemination of human bacterial pathogens, including their transmission patterns, risk factors for and control of infectious disease in human populations Clonal complex: A group of bacterial isolates showing a high degree of similarity, ideally based on typing methods. Clonal complexes are identical to clonal groups
Isolate A population of bacterial cells in pure culture derived from a single colony. In clinical microbiology, isolates are usually derived from the primary culture of a clinical specimen obtained from an individual patient
Strain A strain may be considered an isolate or group of isolates that can be distinguished from other isolates of the same genus and species by phenotypic and genotypic characteristics Cultures of a particular microorganism, isolated at the same time from multiple body sites of a patient and indistinguishable by typing, also represent a single strain.
Clone Bacterial isolates that, although they may have been cultured independently from different sources in different locations and perhaps at different times, still have so many identical phenotypic and genotypic traits that the most likely explanation for this identity is a common origin within a relevant time span
Outbreak Local, initially small-scale, cluster of disease generally caused by increased frequency of infection in a distinct population (may be caused by single epidemic strains or combinations of different strains)
Typing services for nosocomial pathogens Phenotypic and/or genetic analysis of bacterial isolates, below the species/subspecies level, performed in order to generate strain/clonespecific fingerprints or datasets that can be used, for example, to detect or rule out crossinfections, elucidate bacterial transmission patterns and find reservoirs or sources of infection in humans. Subtyping, a term commonly seen in American literature, is often used as a synonym for typing
REASONS FOR TYPING Surveillance of infectious diseases Study of pathogenesis and the course of infection Outbreak investigation Study of bacterial population genetics Identify emerging pathogenic strains or clones within a species, including potential agents of bioterrorism
Performance criteria Stability This refers to the stability of the markers assessed by the typing method Typeability This refers to a method s ability to assign a type to all isolates tested by it. Discriminatory power This refers to a method s ability to assign a different type to two unrelated strains sampled randomly from the population of a given species. Epidemiological concordance Reproducibility
Convenience criteria Flexibility (or spectrum) Rapidity Accessibility Ease of use Cost Amenability to computerised analysis and incorporation of typing results in electronic databases
Phenotypic typing methods Biotyping Antimicrobial susceptibility testing (antibiogram- based typing) Serotyping Phage and bacteriocin typing SDS-PAGE of cellular and extracellular components Multilocus enzyme electrophoresis (MLEE)
1.Inoclution well 2.L-Arabinose 3.Lactose 4.Melbiose 5.Melezitose 6.Raffinose 7.Inositol 8.Sorbitol 9.Mannitol 10.Gal-lacton 11.Amygdalin 12.Gluconate PhP typing for screening
PhP typing of MRSA
Genotypic typing methods Genotypic typing methods assess variation in the genomes of bacterial isolates with respect to composition presence or absence of plasmids overall structure (e.g., restriction endonuclease profiles, number and positions of repetitive elements precise nucleotide sequence (of one or more genes or intergenic regions).
Fragment-based methods Plasmid typing Restriction fragment length polymorphism (RFLP) methods
Plasmid analysis
Tn1546 like elements digested by ClaI 5kb 3kb M BM4147 1 2 3 4 5 M 2kb 1kb 0.7Kb
RFLP
Hybridization-mediated methods Direct hybridization 1 2 3 4 5 6 170Kb 78 kb M 1 2 3 4 5 6
Ribotyping E. coli rrnb operon intergenic spacer 16S rrna tarn 23S rrna 5S ARN 9 1390 442 1601 2490 Ad rb O4c O16 O24
Ribotype pattern
Genotyping of Salmonella typhi of Iran
Riboprinter It takes only 8 hr to complete a run of riboprinting As compare to 4 days of classical typing
PFGE The chromosome is the most fundamental component of identity One approach has been to digest chromosomal DNA with restriction enzymes Enzymes used to cleave DNA often recognize numerous sites within the bacteria, More recently, restriction enzymes that cleave chromosomal DNA less frequently have been utilized for analysis The resulting DNA fragments are too large to be separated by conventional agarose gel electrophoresis
Two of the most commonly utilized approaches Contour-clamped homogenous electric field (CHEF) Field inversion gel electrophoresis (FISH)
Comparison of Migration: Horizontal vs. CHEF - - - - + + + +
Types Of Pulse Field Gel Electrophoresis Field inversion gel Transverse alternating field Crossed field (Reverse) Contour-clamped homogeneous electric field
CHEF Switch Time Electric Field 1 Electric Field 2 - - - - - - - - + + + + + + + +
PFGE Made it possible to separate large DNA fragments in agarose gels by periodic alternation of the angle of the electric field s direction. These DNA macrorestriction fragments are generated with restriction endonucleases with six or more base pair recognition sites ( rare cutters ) Usually yielding fewer than 30 large fragments, normally ranging in size between 20 and 600 kbp.
Procedure Bacterial growth Plug preparation Cell lysis Lysis buffer (Tris, EDTA, Brij, Pro K, ) Digestion Enterobaceriacea XbaI Enterococci, Staphylococcus, pneumococci SmaI Vibrio NotI,.. Agarose preparation Electrophoresis & staining
PFGE Pulse time Voltage Field Angle Temperature Agarose concentration Buffer concentration
669Kb PFGE Pattern M 1 2 3 4 5 6 7 8 9 10 M 398Kb 310Kb 244Kb 217Kb 173Kb 139Kb 105Kb 76Kb
Interpreting DNA fragment patterns Analytical Software: Gel Compar Thus, genomic pattern similarity values must be based on a sufficiently large number of genomic sites/bands for each isolate
Pulsed Field Gel Electrophoresis (PFGE) Interpretive Criteria 1. Indistinguishable banding pattern same number and size 2. Closely related banding pattern differs due to a single genetic event (usually, 2-3 band difference) 3. Possibly related- banding pattern differs due to 2 independent genetic events (usually, 4-6 band difference) 4. Unrelated banding pattern differs due to changes consistent with 3 genetic events.
E. coli Indistinguishable 2 isolates Closely related 11 isolates Possibly related 4 isolates Unrelated 18 isolates
K. pneumonia Indistinguishable 4 isolates (A/A, B/B) Possibly related 3 isolates Unrelated 9 isolates
P. aeruginosa Closely related 2 isolates Possibly related 6 isolates Unrelated 4 isolates
E. faecalis Possibly related 2 isolates Unrelated 9 isolates
PFGE Pattern of VRE Isolates
PFGE Pattern of Clinical Samples
PFGE Pattern of Urban Sewage
PFGE Pattern of Hospital Sewage
PFGE Pattern of Identical Isolates
MRSA
Pulsed Field Gel Electrophoresis (PFGE) Advantages of PFGE - Highly discriminatory - Reproducible (inter and intra lab) - Adapted to most organisms PFGE has been applied to at least 40 pathogens or pathogen groups CHEF systems have also been used for typing of Candida species - outbreak investigation - tracking evolution or long term spread of pathogens Disadvantages - Initial capital outlay expensive - Requires skilled technologists
COMPARISON AND SELECTION OF First depends on application TECHNIQUES The molecular characterization of nosocomial isolates generates data regarding the interrelatedness of isolates. In an individual patient, the use of molecular characterization can assist in separating relapse from reinfection or, in the case of bacteremia, whether the organism is from the infection or contamination. PFGE is method of choice Plasmid or transposon analysis of strains is used when there is suspicion of dissemination of a particular resistance gene or set of genes
The best method often depends not only on the specific epidemiologic situation but also on the resources available. PFGE analysis provides relatively global chromosomal overview scanning more than 90% of the chromosome (the sum of the restriction fragment sizes) But minor genetic changes may go undetected
As PFGE provides the broadest genomic overview, it likely remains the method of choice as the initial typing method for most epidemiologic investigation. However, on rare occasions, PFGE is unable to resolve discriminatory profiles for certain organisms And other typing methods need to be employed to determine the relatedness of the strains
RECENT ADVANCES IN MOLECULAR EPIDEMIOLOGY: NUCLEOTIDE SEQUENCE-BASED ANALYSIS Variety of molecular typing approaches that focus on either single or multiple chromosomal loci SLST MLST
Looking into the future