Application of Different Typing Methods for Detection of Microbial Contamination of Biological Products and Clean Rooms

Transcription

1 Application of Different Typing Methods for Detection of Microbial Contamination of Biological Products and Clean Rooms Pejvak Khaki Department of Microbiology Razi Vaccine & Serum Research Institute Karaj, Iran

2 What is Typing? The basic premise present in any typing system is that: Epidemiologically related isolates are derived from the clonal expansion of a single precursor And consequently they share characteristics that differ from epidemiologically unrelated isolates Genus species Strain (Clonal isolates) There is sufficient diversity at the species level which enables us to differentiate organisms isolated from Different sources Different times Different geographical regions into subtypes or strains

3 When to apply typing? Why we should typing? What methods should be used? Purpose Testing setting- Facility availability Combination of various typing methods

4 APPLICATION OF TYPING METHODS Characterization of the bacterial strains is essential for epidemiological, clinical purposes and biocontamination of biological products and clean room. In epidemiological terms, the organisms involved in the outbreak or contamination are clonally related i.e. they have a common origin/source. Clonally related organisms are members of the same species. The characterizing (phenotypic and genotypic) of bacterial strains fluctuate overtime both locally and globally.

5 EPIDEMIOLOGICAL STUDY (Contd.) The characterization can increase the level of knowledge about: Strain populations circulating in the Community Temporal and geographical changes among strains The emergence and transmission patterns of certain strains The study of relationship between bacterial isolates can contribute to: Detection of biocontamination of biological products (e.g. vaccine) Assessing the role of Microbial contamination in clean rooms The identification of outbreaks Re-infection/relapse The identification of temporal and geographic changes Forensic applications Monitoring of antibiotic resistance Pathogen distribution-nosocomial nosocomial Control and prevention

6 Moving deeper in the characterization of the pathogen

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10 The microscope serves to visualize bacterial cells The clonoscope serves to visualize the clones composing bacterial populations

11 TYPING METHODS The utility of a particular characteristic of typing is related to its stability within a strain and its diversity within the species. Ideally, a typing method will recognize each unrelated isolate as unique. However, in practice, the technique is considered statistically useful when the index of discrimination for two unrelated isolates is >0.95. > All of typing methods can be evaluated in terms of: Typeability Reproducibility Discriminatory power Ease of performance Ease of interpretation Cost (in terms of time & money)

12 TYPING METHODS (Contd.) There is currently no "gold standard" typing system, against which a new method can be evaluated. Evaluation of a method? Analyzing of epidemic and sporadic isolates and comparing with those of previously well studied approaches

13 TYPING METHODS (Contd.) Several typing methods have been developed: Phenotypic methods Early typing methods, that detect characteristics expressed by the microorganisms Genotypic methods Those that involve direct DNA-based analyses of chromosomal or extrachromosomal genetics elements

14 PHENOTYPIC METHODS Biotyping Serotyping Bacteriophage typing Bacteriocin typing Protein profiling Multilocus enzyme electrophoresis (MLEE)

15 BIOTYPING Initial classification within a number of strains belonging to the species are: Cultural characterizations Biochemical traits Colonial morphology Growth requirements/auxotyping Fermentation ability Carbon source utilization Antimicrobial susceptibility testing: Chromosomal/plasmid

16 SEROTYPING Serotyping is one of the oldest typing procedures (still( useful) Based on reactions with specific antisera against different antigen on the cell surface like LPS, omps, flagella and capsule The method can be applied to many different bacteria Stable Reliable Complex Expensive

17 BACTERIOPHAGE TYPING Bacteriophages (phages), infects bacteria In some strains to lysis In others persist in the bacterial cell (prophage) The method based on sensitivity of the strain to phages Phage typing is highly sensitive The method is used widely for epidemiological studies of numerous bacterial strains worldwide Typing may be Direct; based on direct sensitivity to phages Indirect; based on detection of phages present as prophages in bacteriab Remains the major typing method for Staph.aureus, Pseudomonas aeruginosa, Salmonella.spp and Shigella.spp Complex Different results (various parts of the world) Applicable only for certain species (Non-typeable strains) The method is influenced by genetic mechanisms: Lysogenic conversion, loss of prophages, acquisition or loss of R plasmid

18 BACTERIOCIN TYPING Bacteriocins are bactericidal substances which are active against different bacteria The method is performed by: Testing the sensitivity of bacteria to bacteriocins Testing the ability of production of bacteriocins Stable Labour-intensive Time consuming

19 Protein profiling Is performed by isolating whole cell or cell surface proteins Separating by SDS-PAGE Detection the patterns Depends on polymorphism at protein level Difficulty with interpretation Difficulty in standardization Not used extensively for bacteria More useful for fungi and some viruses

20 Multilocus enzyme electrophoresis (MLEE) Protein profiling Depends on polymorphism at protein level Detects differences in the electrophoretic mobility of soluble metabolic enzymes Detects polymorphism within coded region of gene Difficult to detect mutations at amino acid level Low discriminatory power

21 GENOTYPIC METHODS Plasmid profiling Restriction enzyme based methods: RFLP, PFGE, Ribotyping PCR based methods: RAPD, REP- PCR (e.g.vntr), CFLP, AFLP Sequencing Sequence analysis of single region Multilocus sequence typing (MLST) Sequence analysis of variable repeat region within a gene (as spa typing) Total DNA sequencing DNA microarray Total DNA Different genes

22 PLASMID PROFILING Because problems with typeability, time taken, reproducibility, or discriminatory power have been associated with many phenotypic techniques, numerous systems which use DNA-based methods have been developed Plasmid Analysis is the first DNA based technique that has been successfully applied for epidemiological studies of several bacterial species. Plasmids may be involved in some bacterial characteristics such as antibiotic resistance and virulence factors. In the most basic system, plasmids are isolates and then separated ed electrophoretically in an agarose gel to determine their number and size. More detailed analysis of plasmid can be obtained by digesting the t plasmids with a restriction endonuclease and then comparing the number and size of the resulting restriction fragments. Additional plasmids can also be compared by means of DNA DNA hybridization procedures.

23 PLASMID PROFILING (Contd.) Khaki, et al

24 RESTICTED FRAGMENT LENGTH POLYMORPHISM (RFLP) : Restriction endonucleases cleaves DNA at various sites Generates DNA fragments of varying length RFLP pattern RFLP analysis Differences in the banding pattern represent genetic relatedness Pattern specific of one strain

25 Pulse Field Gel Electrophoresis: First described by Schwartz and Cantor (1984). Often considered as GOLD STANDARD. As compare with conventional agarose gel electrophoresis is in way that the electrical field is applied. Gel electrophoresis is done in an apparatus in which polarity of current is changed at frequent interval. By periodically changing the direction of current large DNA molecules (50 kb- 12Mb) are separated.

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27 RFLP with Probe Principle same as PFGE but ease of: Visualization Interpretation DNA is blotted on membrane, hybridized with known labelled probe

28 RFLP with Probe (Contd.) Probes: rrna; ribotyping (widely used) Toxin production genes (e.g. cholera subunit toxin of vibrio cholerae) Antibiotic degrading enzymes (e.g. mec gene, which encodes methicillin resistance) Insertion sequences (e.g. Tn554, a transposon, of Staphy.aureus or DNA insertion element IS6110 for typing of Mycobacterium tuberculosis isolates) Random chromosomal sequences

29 Ribotyping: RFLP with Probe (Contd.) Ribotyping refers to the use of Southern blot anlysis for detecting polymorphisms that are associated with the ribosomal DNA regions The rrna genes, which are located on the chromosome, are not readily lost, the patterns are not influenced by antibiotic pressure, sure, and the patterns are independent of phage or colicin productions The high degree of conservation of ribosomal RNA genes during evolution among all bacteria allows the use of E.coli RNA probe as a universal probe For organisms with multiple (5-7) ribosomal operons, ribotype patterns typically have bands and provide moderate to good discriminatory power For organisms (Mycobacteria), which have only a single ribosomal operon, ribotyping detects only one or two bands, consequently is of limited utility Ribotyping has been applied to several bacterial species include K.pneumoniae, N.meningitides, H.influenzae, H.ducreyi, Shigella spp, Y.enterocolitica, Leptospira sp., P. multocida, C. trachomatis,

30 RFLP with Probe (Contd.) Advantages: Stability Good discriminatory Reproducibility Typeability Simplifies the interpretation (only a small number of bands ) Uses a commercially available universal probe Disadvantages: Selection of probe most important Labor intensive Time consuming selection of restriction enzyme is crucial Requires large amount of DNA Khaki, et al

31 PCR based methods: RFLP-PCR PCR Locus specific RFLP: The specific locus to be examined is amplified with primers Then digestion with restriction endonuclases And subjected to RFLP analysis Random amplification of polymorphic DNA(RAPD) Also Arbitrary primed PCR (AP-PCR) PCR) Based on the use of short random primers(8-20 bases) That hybridize to the chromosomal DNA arbitrarily at different sites With two cycles at low stringency and many cycles at high stringency (annealing temperature) Generate a discrete set of amplification products

32 Sequencing The basis of all sequencing techniques is the generation of four nested series of labelled DNA fragments. Each fragment with one common end and the other end generated in a specific manner according to one of the four bases. The four set of fragments are separated in parallel by electrophoresis on a high resolution acrylamide gel. Such gels can resolve fragments differing in length by a single nucleotide. The four gel lanes (each corresponding to one of the A, C, G, or T bases) contain a series of labelled bands. Reading upwards from the bottom of the gel gives the sequence of the DNA strand being examined. Several methods are available Chemical sequencing Chain termination sequencing

33 Sequencing (Contd.) Most specific and informative method Standard technique for typing and phylogenetic studies Conserve genes can be sequenced to taxonomy study While less-conserved genes can be used to distinguish between individual related and unrelated strains (typing) The technique can be used directly for sequencing a region of chromosome or used for PCR products Gold standard (esp. for viral typing) Logically best approach but practically not Only a part of chromosome analyzed Labor intensive Expensive Difficult interpretation Time-consuming

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35 DNA microarray Produced by attaching or synthesizing hundreds or thousands of oligonucleotides on a solid support (glass slide, nylon membrane) ) in precise pattern by photolithography DNA microarray technology possesses the advantage of investigating ng many of the genes simultaneously. Highly sensitive and can rapidly read large amount of detailed genetic g information 10 6 different probes /cm3 can be attached Potential uses: Identify simultaneously a range of pathogens,or genetic markers of virulence and antibiotic resistance Used for complete genome sequencing( SARS CoV sequencing in 2003) Used for detection and characterization of biosecurity agents Vaccine development

36 EVALUATION OF THE TYPING TECHNIQUES Typeability Reproducibility Stability Discriminatory Index % Typeability = no.of patterns obtained X 100 no. of isolates types D = 1 [1/N (N 1) Σ nj (nj 1)] Where N : is the total number of stains. S : is the total number of types found and nj : is the number of strains belonging to the jth type. s J=1

37 Data analysis Regardless of the data generated : MLEE (multiple spot pattern), RAPD (single/multiple bands), PFGE (Banding pattern) Each pattern are examined for size (in kilobases) and numbers of DNA fragments Patterns then are screened for different bands Goal is to calculate similarity coefficient (S AB ) S AB values help to make dendogram Various methods: Neighbor joining tree, Unweighted pair group method (UPGM)

38 DENDROGRAM Weight - the rough percentage of all individuals that fall within each cluster Compactness - how similar to one another the elements of a cluster Distinctness - how different one cluster is from its closest neighbor

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41 THANK YOU