Ribotyping article from Cleanroom Technology magazine - Oxoid

CLEANROOM TECHNOLOGY July/August 2006 www.cleanroom-technology.co.uk 21

Fast action is needed in the event ofmicrobial contamination. Colin Booth,vice-president of science andtechnology at Oxoid, explains how newribotyping technology can achieve fasteridentification and corrective action

In many cases of food or pharmaceuticalproduction, contamination problems inthe final product may be the first

indication that there has been a breakdownin process control. Such an event hasenormous financial implications for thecompany as production ceases,contaminated product is destroyed, and timeand resources are ploughed into identifyingand correcting the problem. The longer thistakes, the longer it is until production canresume, with knock-on effects on revenueand demand.

Presented with a final productcontaminant, the first questions to arise are“what is it?” and “where did it come from?”.While the QC laboratory sets aboutanswering these questions, full-scalecleaning and sanitising of the entire

processing area may be initiated. Thisuntargeted approach may be warranted withsome traditional identification methods, butwith the availability of automated“ribotyping” technology it is now possible topinpoint the source of contaminationaccurately, to focus corrective actionprecisely to where it’s needed, and to monitorproactively for problem strains in key areas.

Ribotyping is a method for identifying anisolate by obtaining a genetic fingerprint ofthe bacterial genome that codes forribosomal ribonucleic acid (rRNA).Restriction enzymes are used to cut thehighly conserved rRNA genes, in addition toless conserved flanking genes and intergenicmaterial. The resulting fragments areseparated according to their molecularweight and the resulting pattern, or

fingerprint, is used toidentify the organism bycomparing it toestablished patterns.

It is in the lessconserved, flanking genesand intergenic sections ofthe genome that smallvariations betweenstrains occur. Theinclusion of this material,therefore, allows charact-erisation of the organismto strain level.

Ribotyping offersadvantages over con-ventional phenotypicmethods, since identi-fication is not affected by

culture conditions, growth stages or thestress status of the organism. However,traditionally, ribotyping was a manualmethod, limited primarily to well-equippedspecialised laboratories. It was a time-consuming and labour-intensive method,requiring considerable experience andexpertise.

Furthermore, subjective interpretationand the lack of standardisation resulted in ahigh degree of variation between laboratoriesand even between technicians, dramaticallyreducing the value of performing the testing.

Automated analysisIt wasn’t until an automated ribotypingmethod became available that the fullpotential of this genetic fingerprintingtechnique could be realised.

The DuPont Qualicon RiboPrinterMicrobial Characterisation System (availablein Europe and Australia from Oxoid) is a fullyautomated ribotyping method. Byeliminating the need for manual interventionduring the ribotyping process, this systemremoves the potential for operator error andsubjectivity, providing an easy-to-use,standardised method that allows ribotypingto be performed in the routine QC laboratory.Unlike manual ribotyping and manyphenotypic identification methods, theRiboPrinter System offers same day results.This is an enormous advantage in thepharmaceutical and food productionindustries where delays are costly.

Pure culture samples are added to thesystem following heat treatment and theaddition of a lysing agent. Cell lysis releasesthe bacterial DNA (see figure 1). The systemthen processes the samples automatically,performing the following steps:

C O N T A M I N A T I O N I D

Picking samples

Membrane processingPattern detection

Data processing

Separation & transfer

Heat treatment DNA preparation

Well

Gel

DNA fragmenttravel

DNAfragments

AnodeMembrane

Membrane travel

RIBOPRINT

External to Riboprinter system

Figure 1: Samplepreparation and analysis

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Pinpointingmicrobial contamination

Ribotyping has been automated,giving faster results

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I D E N T I F I C A T I O N

22 www.cleanroom-technology.co.uk July/August 2006 CLEANROOM TECHNOLOGY

■ DNA fragmentation using restrictionenzymes. The primary enzyme used by theRiboPrinter system is EcoR1. Pvu II is alsosupplied for Micrococcus, Kocuria,Pseudomonas and Salmonella. Laboratoriesalso have the flexibility to use otherrestriction enzyme protocols, if necessary.■ Separation of fragments according to theirsize by gel elctrophoresis■ Transfer of fragments to a movingmembrane■ Hybridisation of the fragment bands,causing key fragments to chemiluminesce■ Capture of the resulting pattern, orRiboPrint image, using a low-light camera,followed by storage of the digitised image ■ Data analysis compares the RiboPrintpattern against those in the integral DuPontIdentification Database (or the user’s owndatabase) to produce a characterisation oridentification report.

This automated ribotyping method canprocess eight isolates within just eight hours.In addition, new batches can be started everytwo hours, enabling as many as 32 isolates tobe loaded in a normal working day.

Species identificationAs soon as a RiboPrint pattern is obtained,the system compares it to the patterns storedin the integral database. This powerfuldatabase contains more than 6400 patterns,from some 200 genera and more than 1400species that are of special interest to thepharmaceutical and food industries.Proprietary identification algorithms usefragment size, the number of bands andsignal intensity to assign a definitive species-level identification.

If the isolate cannot be identified using theintegral database, it may still be possible toobtain an identification by comparing thepattern to the user’s Custom IdentificationDatabase (populated with data from localstrains and externally generatedidentifications).

Independently of the identificationprocess, the RiboPrinter Systemautomatically characterises each isolate atthe substrain level. Each RiboPrint pattern isplaced in a RiboGroup, which is a set ofstatistically identical patterns. If the softwarerecognises a new pattern asindistinguishable from anexisting pattern in thedatabase, it assigns that newsample to the sameRiboGroup. However, if thepattern does not match anexisting RiboGroup, it isstored as a new RiboGroup.

Each time a new sample isadded to an existingRiboGroup, the system

software incorporates thesample’s pattern data into anew composite patternrepresentative of the entiregroup. Subsequent patternsare then compared againstthe new RiboGroup. TheRiboGroup library isdynamic, changing as moreisolates are analysed.

This sub-species charact-erisation allows companies topinpoint a source ofcontamination with greaterspeed, ease and precisionthan was possible before.

Food sector case studyStaphylococcus contamination was found ina ready-to-eat food product.1 Conventionalmethods identified the culprit asStaphylococcus epidermidis. However, thisspecies was found to be present in manyareas around the production site. Potentially,this could have required the entire site to becleaned and sanitised. Each isolate wasanalysed using the automated RiboPrintersystem. By comparing the RiboGrouppatterns, only one isolate was found to be thesame strain as the end product contaminant.The source was identified to be the hands ofone employee. Simple and effectivemeasures were implemented to correct theproblem, avoiding costly closure of the siteand further reduction in output.

Pharma sector case studyA bacterial contaminant was found in anasthma inhaler formulation during the finalstages of clinical trials.2 Phenotypic methodsfailed to identify the organism or to find thesource of contamination. The trial was haltedand each day that went by increased thedelay in bringing the product to market.

Turning to the RiboPrinter System for help,within eight hours the culprit was identifiedas Enterobacter cloacae – an organism thathad dangerous implications for the targetusers of the product. Several different strainsof E. cloacae were found in several of the raw

materials, but only oneof these strains matchedthe contaminant in thefinal product. Thesource of the problemwas found to be theinert carrier for theactive ingredient. Oncethe company switchedto a higher gradeproduct, the problem

was resolved and trials could be resumed.FDA-regulated industries must ensure that

any system that stores data is compliant withthe Code of Federal Regulations forelectronic record security (21 CFR Part 11).The Windows-based RiboPrinter softwaremeets this requirement with four levels ofsecurity that determine user access to certainfeatures, along with detailed audit trails totrack and record any changes to data.

In addition to answering the importantquestions, “What is it?” and “Where did itcome from?”, with speed and precision, theautomated ribotyping method also answersthe question, “Have we seen it before?”.

Each pattern, or fingerprint, is linked tohistorical data, so that, should a problemorganism recur, operators can find outquickly where and when it appeared before.Historical mapping and trend analyses thenallow manufacturers to gain a greaterunderstanding of the microbial environmentwithin their facilities. Such knowledgeenables them to take a proactive approach inthe control of the aseptic environment,allowing them to anticipate problems beforethey lead to interruptions in production.

The microbial characterisation that ismade possible by automated ribotyping,means that, even in the unlikely event that anidentification cannot be obtained, the sourceof contamination can still be found andeliminated. This precise pinpointing of thesource of contamination allows moretargeted action and ensures that productionis resumed as quickly as possible. ■

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CONTACTOxoid LimitedWade RoadBasingstokeHampshire RG24 8PW, UKT +44 1256 841144F +44 1256 463388oxoid@oxoid.comwww.oxoid.com

References:

1. Campden and Chorleywood Food ResearchAssociation Newsletter, January 2000.2. Drug Developers can't leave identification ofpotentially lethal organisms to chance. RiboPrinterCharacterisation System Application Profile. Availablefrom Oxoid Limited.

2 DPBETA7 102–69–S–1

4 DPBETA7 102–69–S–3

5 DPBETA7 102–69–S–5

2 DPBETA7 102–69–S–1

4 DPBETA7 102–69–S–3

5 DPBETA7 102–69–S–5

2 DPBETA7 102–69–S–1

4 DPBETA7 102–69–S–3

5 DPBETA7 102–69–S–5

Grayscale RiboPrint® patterns

Waveform RiboPrint® patterns

Dual graphics RiboPrint® patterns

Figure 2: The RiboPrint patternfor a bacterial isolate

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