microbial identification: the keys to a successful program

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Microbial Identification:The Keys to a Successful

Program

Mary Griffin and Dona ReberEditors

PDABethesda, MD, USADHI Publishing, LLCRiver Grove, IL, USA

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ISBN: 1-933722-65-7Copyright © 2012 Mary Griffin and Dona ReberAll rights reserved.

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CONTENTS

FOREWORD xvDennis Guilfoyle

PREFACE xix

1 REGULATORYAND COMPENDIA GUIDANCEON MICROBIAL IDENTIFICATIONS INCLUDINGRECENT REGULATORY FINDINGS 1Anthony M. CundellIntroduction 1Regulatory Guidelines 2Compendial Guidance 6Microbial Identification as an Issue in FDAWarning Letters 13Conclusions 14References 14About the Author 15

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2 PRIMARY IDENTIFICATION METHODS:BACK TO BASICS 17Marsha Stabler HardimanCulture Media 18Temperature Conditions 23Culture Characteristics 23Pure Culture Isolation 25Gram Stain 27Spore Stain 30Microscopy 30Biochemical Screening Tests 31

Oxidase test 32Catalase test 32Coagulase test 33Multitest phenotypic systems 34API strips 34Enterotube 35Training 35

Conclusion 36References and Further Reading 37About the Author 38

3 MICROBIAL IDENTIFICATIONS INTHE QUALITY CONTROL LABORATORY:A MULTI-COMPONENTAPPROACH 39Scott SuttonIntroduction 39Why do we Identify Microorganisms in QC Microbiology? 40

Internal QC of cultures 40Tracking/trending 41

Raw material/API testing (including water asa raw material) 41Non-sterile pharma and personal productstesting — microbial limits and “absence of ...” 41Routine environmental monitoring 42Gram stain — people/soil/water 44Genus–species 45Environmental monitoring excursions 45

Product failures/issues 45Microbial Identification TechnologiesPerformance Requirements for Identification Technologies 46Polyphasic Identification Strategies 46

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What is polyphasic identification? 46Polyphasic identification and the concept of “species” 47Polyphasic identification and the QC microbiologylaboratory 47Case studies 48

Case study #1 — Burkholderia cepacia 48Case study #2 — Bacillus subtilis, B. cereus, etc. 49

Performance Requirements for Identification Technologies 50What are the Popular Methods for Microbial Identification

in QC Microbiology? 52Phenotypic 52

Gram stain/microscopy 53Selective and differential media 54Test tubes 54API Strips 55BBL Crystal ID System 56Vitek® 56Biolog® 57MIDI-Sherlock® 58

Genotypic 59MicroSeq® 59Riboprinter® 60Bacterial barcodes 60

Proteotypic — MALDI-TOF 61Other options 61

Conclusions 62References 63About the Author 68

4 CHALLENGES IN MICROBIAL IDENTIFICATIONS 69Jeanne MoldenhauerBackground on Microbial Identifications 69Selecting the System for Use 71How to Distinguish between Systems for Specific

Types of Organisms 74Taxonomic Issues 76Typical Problems in Identifications 79

Organism preparation for testing 80Use of similarity numbers 81Phenotypic methodsRiboPrinter® methods 81Verification of microbial identification methods 83

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Difficulties in identification 83Making the right assumptions 83

Conclusions 84References 84About the Author 86

5 VALIDATION OF AN AUTOMATED MICROBIALIDENTIFICATION SYSTEM 87Amy McDanielIntroduction 87SystemValidation: Initial Considerations 88Validation Plan 90Assessments 92User Requirements Specification 94Installation, Operational and Performance Qualifications 96Traceability Matrices 103Instrument SOPs 103Summary Reports 104Conclusion 104References 105About the Author 106

6 FUNGAL IDENTIFICATION 107Ziva AbrahamMold Contamination 107Nature of Fungi 110Classification 111

Zygomycetes 111Ascomycetes 112Basiodiomycetes 113Deuteromycetes 113

Anamorphs and Teleomorphs 114Medically Important Fungi 115Clinical Importance of Zygomycetes 116Clinical Importance of Ascomycetes 116Clinical Importance of Deuteromycetes 116

Dimorphic pathogens 117Yeasts 118

Identification Basics 118Growth and Sporulation Media 119Preparation for Microscopic Examination of Fungi 120

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Role of the Stain 120Tease Mount Technique 121

Tape lift technique 121Slide culture method 122

Microscopy 122Stereo microscope 122Using a compound microscope 123

Identification Keys 124Appearance of growth 124Examining the color of colony and color on reverseof the plate 124Special features in surface growth 124Texture of growth 125Observation under the microscope 125

Zygomycetes 125Ascomycetes 128Deuteromycetes 131

Hylaine hyphae 131Pigmented (dematiaceous) hyphae 134

Automation in Fungal Identification 136Phenotypic technology 136

Genotypic Technology 138Matrix Assisted Laser Desorption Ionization–Time of Flight

(MALDI-TOF) Mass Spectrometry 139Beta Glucans 140Mycotoxins 142

Safety Precautions in Laboratory Operations 144Conclusion 146References 147About the Author 150

7 IDENTIFICATION OFADVENTITIOUSVIRAL CONTAMINANTS OF CELLCULTURE PROCESSES 151Martina Kopp and Houman DehghaniIntroduction 151Viral Contamination of Cell Culture — Perspective 153Contamination of Cell Culture with MMV 154Mouse MinuteVirus 156Contamination of Cell Culture with Vesivirus 157Vesiviruses 158Evaluation Process for Confirmation and Identification

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of a Viral Contamination 161In vitro Adventitious Virus Assay 164Nucleic Acid Based Methods for Detection and Identification

of Viruses 167Emerging Nucleic Acid Based Technologies for Virus Identification 171Regulatory Aspects 173Conclusions 174References 175About the Authors 183

8 IDENTIFYING MYCOPLASMA CONTAMINATION:CONCEPTS AND TOOLS 185Shayn E.Armstrong, Jill A. Mariano, Cynthia A. Martino, and John A. RyanIntroduction 185Taxonomy and Biology of Mollicutes 186Incidence 189

Sources and transmission 190Raw material filtration and irradiation 192

Regulations 194Current Technologies 196

Surveillance testing 196In-process testing 197Final product testing 197Where to test 198Direct methods 198Indirect methods 200

Indicator cell culture procedure 200Test material qualification 202Mycoplasma elimination 203

RMM for Detecting Mycoplasma 205When to consider a RMM 205Pros and cons 205RMM suitability 206Development and optimization of a PCR-based RMM 207

Conclusion 212References 213About the Authors 219

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9 MICROBIAL IDENTIFICATIONSINTHE COSMETICS INDUSTRY 221Donald J. EnglishIntroduction 221Microbiological Testing in the Cosmetic Industry 222

Quality control testing of microbial strains, growthpromotion, reagents and identification kits 222Microbial content testing 223

Raw ingredients and cosmetic products susceptibleto microbial contamination 223Raw ingredients and cosmetic products notsusceptible to microbial contamination 224

Preservative challenge testing 226In-use testing 227Cleaning and sanitization 228Environmental testing 229

Identification of Microbial Isolates 230Bacteria and yeast isolates 230

Gram-positive bacilli 231Gram-positive cocci 237Gram-negative bacilli 244Yeast 249

Mold 253Summary 254References and Further Reading 255About the Author 268

10 MICROBIAL IDENTIFICATIONS INBIOPHARMACEUTICAL MANUFACTURING 271Evelyn Der and Carole GenovesiSample Types Processed for Identification 272

Raw materials (ingredients and excipients) 272Drug substance 272Drug product and active pharmaceutical ingredients 273Disinfectants 273Process simulations 274Sterility testing 275Culture collection/cell banks 275Biological indicators 276Water 276Environmental monitoring 277Gases 277

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Identification Systems 278Phenotypic 278

BioMérieux API® System 278BioMérieuxVitek® 2 System 279MIDI Sherlock® 280

Genotypic 281Applied Biosystem MicroSeq® System 281DuPont Qualicon RiboPrinter® System 282

Outsourcing identifications 283Typical biopharmaceutical identification process flow 284Important considerations for the currently availableID processes 284

Disposition of Recovered Isolates 287Typical isolates recovered 289Treatment of novel organisms 291Retention and use of retained isolates 291

Microbial Identification Trending 293Trending groups 293

Gram positive coccus 294Gram positive rods nonspore-formers 294Gram positive rods spore-formers 294Gram negative rods non-fermenters 295Gram negative rods fermenters 295Yeasts and molds 296

Trending frequency and evaluation 296Tools for trending 297

Laboratory Information Management Systems (LIMS) 297Interpretation of trends 299

Conclusion 299References 300About the Authors 302Appendix A1 Example of Microbial Trending — Partial LIMS Report 303Appendix A2 Example of Microbial Trending – Excel Graph

Created from LIMS Report 304Appendix A3 Quarterly Microbial Identification Trend Evaluation 306

11 MICROBIAL IDENTIFICATION —A MEDICAL DEVICE PERSPECTIVE 307Nina McAuliffeImportant Regulatory Requirements 308Current Regulatory Trends 312Contamination Risk Assessment 313

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Technology as a Role Player 314KnowingYour Microbiome 315Utilizing Your Data 316Basic Examples 317Summary 319References 321About the Author 322

12 CONSTRUCTINGA MICROBIALIDENTIFICATION LABORATORY: KEYCOMPONENTS AND ELEMENT BENEFITS 323Mary J. GriffinIntroduction 323Physical Layout Component 324

Physical Layout: safety element considerations 326Physical Layout: efficiency element considerations 327

Organization Function Component 329Organization Function: quality/complianceOrganization Function: safety element considerations 330Organization Function: efficiency element considerations 330

Routine Operations Component 331Routine Operations: quality/compliance elementconsiderations 332

Stock culture program 332Routine Operations: safety element considerations 333Routine Operations: efficiency element considerations 333

Centralized microbial testing laboratory 334Laboratory qualification 334

Conclusion 335References 344About the Author 344

13 DETERMINATION OF OBJECTIONABLEORGANISMS IN NON-STERILEPHARMACEUTICAL PRODUCTS —A SCIENCE-BASED RISKASSESSMENTAPPROACH 347Rhonda EzellEmerging Issues Regarding Objectionable Microorganisms

in Non-Sterile Pharmaceutical Products 348The Changing Patient Population 352

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Warning Letters and Form 483 Observations 353Definition of Objectionable Microorganisms 356Information from the FDA and the USP 357Who Should DetermineWhether a Microorganism

is Objectionable? 359The Risk Assessment 360Conclusions 364References 366About the Author 368

14 LOOKINGTOTHE FUTURE: RAPIDANDAUTOMATED MICROBIAL IDENTIFICATIONTECHNOLOGIES 369Michael J. MillerIntroduction 369A Brief History Lesson 370Rapid and Automated Microbiological Technologies 371Growth-Based ID and Presence/Absence Technologies 375

Utilization of biochemical and carbohydrate substratesfor microbial identification 375Use of selective media for the rapid and automateddetection of specific microorganisms 377

Cellular Component-Based ID and Presence/AbsenceTechnologies 377

Fatty acid analysis for microbial identification 377MALDI-TOF mass spectrometry for microbial identification 378SELDI-TOF mass spectrometry for microbial identification 378Fourier Transform–Infrared (FT-IR) spectrometry formicrobial identification 379

Optical Spectroscopic-Based ID and Presence/AbsenceTechnologies 379Elastic scattering for the detection of specific microorganisms 380Inelastic scattering for the detection of specificmicroorganisms 381

Nucleic Acid Amplification-based ID and Presence/AbsenceTechnologies 382Ribotyping for bacterial identification and straindifferentiation 383PCR for the detection of specific microorganisms 384SYBR® Green and Taqman® probes 385MALTI-TOF mass spectrometry of PCR productsfor microbial identification 388

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Electrospray TOF mass spectrometry of PCRproducts for microbial identification 388Gene sequencing for microbial identification 389Detection of mycoplasma 390

MEMS-Based ID and Presence/Absence Technologies 392Microfluidics or Lab-on-a-Chip Systems formicrobial identification 392Microarrays for microbial identification of mycoplasma 393Micro and nanocantilevers for microbial detection 394

Summary 395References 395About the Author 396

15 IMPLICATIONS OF THE HUMANMICROBIOME PROJECT TOPHARMACEUTICAL MICROBIOLOGY 399Anthony M. CundellIntroduction 399Implications to Pharmaceutical Microbiology 401Conclusions 404References 404About the Author 405

16 MICROBIAL KNOWLEDGE MANAGEMENT 407Frank HallinanIntroduction 407

ICH Q10 and knowledge management 408KM and environmental microbiology 409

Microbiological Data Challenges 410ICH Q10 and KM 412

Conclusions 419Summary 420References 420About the Author 421

Index 423

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FOREWORD

My excitement about the publication of Microbial Identifications: TheKeys to a Successful Program resonates with me on several levels. Thefirst fulfills a need as an FDA regulatory microbiologist and thesecond as a university professor. Finally, the authors’ contributionsare vital because they are internationally recognized experts in theirrespective microbiological niches. Many of these individuals arepersonal friends of mine, and I have collaborated professionally withmany of them for over 30 years. My respect for their credentials andcommunicative abilities is unqualified. For anyone who pursues acareer in the pharmaceutical, medical device, cosmetic, biotech-nology or related health care industries; and those who participate inthe regulations of these industries this publication is a must-read.

Those of us who are career industry/government/academiamicrobiologists are well aware of numerous other publicationsthat address the topic of Microbial Identification. Although oneshould not minimize their contribution to the fields of microbiologyand healthcare, the difference between these preexisting referencesand the contents of Microbial Identifications: The Keys to a SuccessfulProgram, should be made clear. The defined chapters in this book

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present a well-balanced scientific rationale for the application ofcurrent technology together with personal experience/experimentsin conjunction with data derived from these classic textbooks andpeer review literature. The synergism of sound fundamentalmicrobiology, emerging contemporary instrumentation and theapplication of today’s risk-assessment priority will label this workas a new benchmark for literature excellence in pharmaceuticalmicrobiology.

The ubiquitous presence of microorganisms, whether bacteria,fungi or viruses, have been the cause of contamination in people,processes and products throughout time. The need to identify andtrack the specific species when these outbreaks occur is critical forthe epidemiology and trace-back activities associated with locatingthe microbial source in an effort to remediate the cause. The rangeof topics covered in the 16 chapters of this reference book will makethe task unequivocally easier when investigating microbialproblems and proposing pragmatic solutions or explanations. Theauthors are insightful, comprehensive and balanced in theirprospective viewpoints and allow the reader the full availability ofadditional citations in support of their positions. This book isuniquely written in order to assist with the understanding andapplication of “The Keys” to address the microbial identificationcomponent for the many industrial and governmentresponsibilities performed by pharmaceutical microbiologists.Individual scientific facts are always important, but detailed advicefrom experts, with decades of experience, is priceless.

The regulatory and industrial application of this book cannot beoveremphasized. There has been a global realization regarding theimportance of microbiological identification by governments andstandard setting institutions. The risk assessment of microbialintrusion on a wide range of consumer products (or theirmanufacturing environments and components), both sterile and non-sterile, has become codified in regulatory requirements as well ascompendial standards.Microbial Identifications: The Keys to a SuccessfulProgram clearly and effectively describes these changes so that thereader is not caught unprepared to meet the paradigm shift that hasemerged from both domestic and international cGMP regulations.

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Perhaps one may be confronted with the need to purchase anew rapid micro method (RMM) platform for microbialidentification. The choices are numerous. Consequently, this makesselecting the best RMM for your purposes all the more challenging.The principles and benefits of most RMMs are clearly described inthis book. The reader gets real solutions to the essential questions of“Why, When and How” when making these important decisions. Iassure you that the reader will find an appropriate chapter toaddress their microbiological quandary.

I am grateful for the opportunity to write the foreword to thistext book. Its contents will be beneficial to a multitude of readers:inclusive of industrial/government/academic microbiologists,quality assurance and control, risk management and decision-making personnel.

Dennis E. Guilfoyle, Ph.D.Pharmaceutical MicrobiologistNortheast Regional Laboratory

US Food and Drug Administration

Disclaimer: These comments are those of the author only and do not necessarilyrepresent the positions of the FDA.

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microbial identification: the keys to a successful program

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