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Library of Congress Cataloging-in-Publication Data
Names: Wooley, Dawn P., editor. | Byers, Karen B., editor.
Title: Biological safety : principles and practices / edited by Dawn P. Wooley, Department of
Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, OH, Karen B. Byers,
Dana Farber Cancer Institute, Boston, MA.
Description: 5th edition. | Washington, DC : ASM Press, [2017] | Includes index.
Identifiers: LCCN 2017000395 (print) | LCCN 2017004110 (ebook) |
ISBN 9781555816209 (print) | ISBN 9781555819637 (ebook)
Subjects: LCSH: Microbiological laboratories—Safety measures. | Biological laboratories—Safety measures.
Classification: LCC QR64.7 .L33 2017 (print) | LCC QR64.7 (ebook) | DDC 570.289—dc23
LC record available at https://lccn.loc.gov/2017000395
doi:10.1128/9781555819637
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, USA.
Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, USA.
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Biological safety principles and practices Byers
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- 4. Biological Safety 5TH EDITION PRINCIPLES AND PRACTICES DAWNWOOLEY AND KAREN BYERS
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- 7. Safety 5TH EDITION PRINCIPLES AND PRACTICES DAWNP. WOOLEY Wright State University, Dayton, Ohio KAREN B. BYERS Dana Farber Cancer Institute, Boston, Massachusetts EDITED BY Biological Washington, DC
- 8. Copyright © 2017by ASM Press. ASM Press is a registered trademark of the American Society for Microbiol- ogy. All rights reserved. No part of this publication may be reproduced or transmitted in whole or in part or reutilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Disclaimer: To the best of the publisher’s knowledge, this publication provides information concerning the subject matter covered that is accurate as of the date of publication. The publisher is not providing legal, medical, or other professional services. Any reference herein to any specific commercial products, procedures, or services by trade name, trademark, manufacturer, or otherwise does not constitute or imply endorsement, recommendation, or favored status by the American Society for Microbiology (ASM). The views and opinions of the author(s) expressed in this publication do not necessarily state or reflect those of ASM, and they shall not be used to advertise or endorse any product. Library of Congress Cataloging-in-Publication Data Names: Wooley, Dawn P., editor. | Byers, Karen B., editor. Title: Biological safety : principles and practices / edited by Dawn P. Wooley, Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, OH, Karen B. Byers, Dana Farber Cancer Institute, Boston, MA. Description: 5th edition. | Washington, DC : ASM Press, [2017] | Includes index. Identifiers: LCCN 2017000395 (print) | LCCN 2017004110 (ebook) | ISBN 9781555816209 (print) | ISBN 9781555819637 (ebook) Subjects: LCSH: Microbiological laboratories—Safety measures. | Biological laboratories—Safety measures. Classification: LCC QR64.7 .L33 2017 (print) | LCC QR64.7 (ebook) | DDC 570.289—dc23 LC record available at https://lccn.loc.gov/2017000395 doi:10.1128/9781555819637 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1 Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, USA. Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, USA. Phone: 800-546-2416; 703-661-1593. Fax: 703-661-1501. E-mail: books@asmusa.org Online: http://www.asmscience.org
- 9. Contents INTRODUCTION Contributors ix Foreword—Caryl P.Griffin and James Welch xiii Preface xv SECTION I. HAZARD IDENTIFICATION 1. The Microbiota of Humans and Microbial Virulence Factors 3 Paul A. Granato 2. Indigenous Zoonotic Agents of Research Animals 19 Lon V. Kendall 3. Biological Safety Considerations for Plant Pathogens and Plant-Associated Microorganisms of Significance to Human Health 39 Anne K. Vidaver, Sue A. Tolin, and Patricia Lambrecht 4. Laboratory-Associated Infections 59 Karen Brandt Byers and A. Lynn Harding SECTION II. HAZARD ASSESSMENT 5. Risk Assessment of Biological Hazards 95 Dawn P. Wooley and Diane O. Fleming 6. Protozoa and Helminths 105 Barbara L. Herwaldt
- 10. vi | CONTENTS 7. Mycotic Agents147 Wiley A. Schell 8. Bacterial Pathogens 163 Travis R. McCarthy, Ami A. Patel, Paul E. Anderson, and Deborah M. Anderson 9. Viral Agents of Human Disease: Biosafety Concerns 187 Michelle Rozo, James Lawler, and Jason Paragas 10. Emerging Considerations in Virus-Based Gene Transfer Systems 221 J. Patrick Condreay, Thomas A. Kost, and Claudia A. Mickelson 11. Biological Toxins: Safety and Science 247 Joseph P. Kozlovac and Robert J. Hawley 12. Molecular Agents 269 Dawn P. Wooley 13. Biosafety for Microorganisms Transmitted by the Airborne Route 285 Michael A. Pentella 14. Cell Lines: Applications and Biosafety 299 Glyn N. Stacey and J. Ross Hawkins 15. Allergens of Animal and Biological Systems 327 Wanda Phipatanakul and Robert A. Wood SECTION III. HAZARD CONTROL 16. Design of Biomedical Laboratory and Specialized Biocontainment Facilities 343 Jonathan T. Crane and Jonathan Y. Richmond 17. Primary Barriers and Equipment-Associated Hazards 367 Elizabeth Gilman Duane and Richard C. Fink 18. Primary Barriers: Biological Safety Cabinets, Fume Hoods, and Glove Boxes 375 David C. Eagleson, Kara F. Held, Lance Gaudette, Charles W. Quint, Jr., and David G. Stuart 19. Arthropod Vector Biocontainment 399 Dana L. Vanlandingham, Stephen Higgs, and Yan-Jang S. Huang 20. Aerosols in the Microbiology Laboratory 411 Clare Shieber, Simon Parks, and Allan Bennett 21. Personal Respiratory Protection 425 Nicole Vars McCullough 22. Standard Precautions for Handling Human Fluids, Tissues, and Cells 443 Debra L. Hunt 23. Decontamination in the Microbiology Laboratory 463 Matthew J. Arduino 24. Packing and Shipping Biological Materials 475 Ryan F. Relich and James W. Snyder
- 11. CONTENTS | vii SECTION IV. ADMINISTRATIVECONTROL 25. Developing a Biorisk Management Program To Support Biorisk Management Culture 495 LouAnn C. Burnett 26. Occupational Medicine in a Biomedical Research Setting 511 James M. Schmitt 27. Measuring Biosafety Program Effectiveness 519 Janet S. Peterson and Melissa A. Morland 28. A "One-Safe" Approach: Continuous Safety Training Initiatives 537 Sean G. Kaufman 29. Biosafety and Biosecurity: Regulatory Impact 551 Robert J. Hawley and Theresa D. Bell Toms SECTION V. SPECIAL ENVIRONMENTS 30. Biological Safety and Security in Teaching Laboratories 565 Christopher J. Woolverton and Abbey K. Woolverton 31. Biosafety in the Pharmaceutical Industry 585 Brian R. Petuch 32. Biosafety Considerations for Large-Scale Processes 597 Mary L. Cipriano, Marian Downing, and Brian R. Petuch 33. Veterinary Diagnostic Laboratories and Necropsy 619 Timothy Baszler and Tanya Graham 34. Special Considerations for Animal Agriculture Pathogen Biosafety 647 Robert A. Heckert, Joseph P. Kozlovac, and John T. Balog 35. Biosafety of Plant Research in Greenhouses and Other Specialized Containment Facilities 665 Dann Adair, Sue Tolin, Anne K. Vidaver, and Ruth Irwin 36. Biosafety Guidelines for Working with Small Mammals in a Field Environment 679 Darin S. Carroll, Danielle Tack, and Charles H. Calisher 37. Components of a Biosafety Program for a Clinical Laboratory 687 Michael A. Pentella 38. Safety Considerations in the Biosafety Level 4 Maximum-Containment Laboratory 695 David S. Bressler and Robert J. Hawley Index 719
- 13. Contributors Dann Adair Conviron, Pembina,North Dakota Deborah M. Anderson Laboratory for Infectious Disease Research and Depart- ment of Veterinary Pathobiology, University of Missouri, Columbia, Missouri Paul E. Anderson Laboratory for Infectious Disease Research and Depart- ment of Veterinary Pathobiology, University of Missouri, Columbia, Missouri Matthew J. Arduino Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia John T. Balog U.S. Food and Drug Administration, Office of Operations, Employee Safety and Environmental Management, Silver Spring, Maryland Timothy Baszler Washington State University, Paul G. Allen School for Global Animal Health, Pullman, Washington Allan Bennett Public Health England, Biosafety, Porton, Salisbury, Wiltshire, United Kingdom David S. Bressler Centers for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia LouAnn C. Burnett International Biological and Chemical Threat Reduction, Sandia National Laboratories, Albuquerque, New Mexico Karen Brandt Byers Dana Farber Cancer Institute, Boston, Massachusetts Charles H. Calisher Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado Darin S. Carroll Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia Mary L. Cipriano Abbott Laboratories, North Chicago, Illinois (retired) J. Patrick Condreay pc Biosafety Consulting Services, LLC, Carrboro, North Carolina ix
- 14. x | CONTRIBUTORS Jonathan T. Crane HDR,Inc., Atlanta, Georgia Marian Downing Abbott Laboratories, North Chicago, Illinois (retired) Elizabeth Gilman Duane Environmental Health and Engineering Inc., Needham, Massachusetts David C. Eagleson The Baker Company, Inc., Sanford, Maine Richard C. Fink Environmental Health and Engineering Inc., Needham, Massachusetts, and Pfizer (retired) Diane O. Fleming Biological Safety Professional (retired), Mitchellville, Maryland Lance Gaudette The Baker Company, Inc., Sanford, Maine Tanya Graham Biosafety Consulting for Veterinary Medicine, LLC, Esteline, South Dakota Paul A. Granato Department of Pathology, SUNY Upstate Medical University, Syracuse, New York, and Laboratory Alliance of Central New York, LLC, Liverpool, New York A. Lynn Harding Biosafety Consultant, Chattanooga, Tennessee J. Ross Hawkins Division of Advanced Therapies, National Institute for Biological Standards and Control a centre of the Medicines and Healthcare Regulatory Agency, South Mimms, Herts, United Kingdom Robert J. Hawley Consultant, Biological Safety and Security, Frederick, Maryland Robert A. Heckert Robert Heckert Consulting, Palm Desert, California Kara F. Held The Baker Company, Inc., Sanford, Maine Barbara L. Herwaldt Centers for Disease Control and Prevention, Parasitic Diseases Branch, Atlanta, Georgia Stephen Higgs Biosecurity Research Institute, Kansas State University, Manhattan, Kansas Yan-Jang S. Huang Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas Debra L. Hunt Duke University, Durham, North Carolina Ruth Irwin Information Systems for Biotechnology, Virginia Polytechnic Institute & State University, Blacksburg, Virginia Sean G. Kaufman Behavioral-Based Improvement Solutions, Woodstock, Georgia Lon V. Kendall Department of Microbiology, Immunology and Pathology and Laboratory Animal Resources, Colorado State University, Fort Collins, Colorado Thomas A. Kost GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina (retired) Joseph P. Kozlovac USDA ARS Office of National Programs, Animal Production & Protection, Beltsville, Maryland Patricia Lambrecht Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, Nebraska James Lawler Navy Medical Research Center, Clinical Research, Fort Detrick, Maryland Travis R. McCarthy Laboratory for Infectious Disease Research, University of Missouri, Columbia, Missouri Nicole Vars McCullough 3M, Personal Safety Division, Saint Paul, Minnesota Claudia A. Mickelson EHS Office, Massachusetts Institute of Technology, Cambridge, Massachusetts (retired) Melissa A. Morland University of Maryland, Baltimore, Baltimore, Maryland Jason Paragas Lawrence Livermore National Laboratory, Global Security, Livermore, California Simon Parks Biosafety, Air and Water Microbiology Group, Public Health England, Porton Down, Wiltshire, United Kingdom
- 15. CONTRIBUTORS | xi Ami A. Patel Laboratoryfor Infectious Disease Research, University of Missouri, Columbia, Missouri Michael A. Pentella Massachusetts Department of Public Health, State Public Health Laboratory, Jamaica Plain, Massachusetts Janet S. Peterson Biosafety Consultant, Ellicott City, Maryland Brian R. Petuch Global Safety & Environment, Merck, West Point, Pennsylvania Wanda Phipatanakul Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts Charles W. Quint, Jr. The Baker Company, Inc., PO Sanford, Maine Ryan F. Relich Division of Clinical Microbiology, Indiana University Health Pathology Laboratory, and Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana Jonathan Y. Richmond Bsafe.us, Southport, North Carolina Michelle Rozo Navy Medical Research Center, Clinical Research, Fort Detrick, Maryland Wiley A. Schell Department of Medicine, Division of Infectious Diseases and International Health, Duke University, Durham, North Carolina James M. Schmitt Occupational Medical Service, National Institutes of Health, Bethesda, Maryland Clare Shieber Public Health England, Biosafety, Air and Water Microbiology Group, Porton, Salisbury, Wiltshire, United Kingdom James W. Snyder Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky Glyn N. Stacey Division of Advanced Therapies, National Institute for Biological Standards and Control a centre of the Medicines and Healthcare Regulatory Agency, Blanche Lane, South Mimms, Herts, United Kingdom David G. Stuart The John M. Eagleson, Jr. Institute, Kennebunk, Maine Danielle Tack Poxvirus and Rabies Branch, Centers for Disease Control and Prevention, Atlanta, Georgia Sue A. Tolin Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute & State University, Blacksburg, Virginia Theresa D. Bell Toms Leidos Biomedical Research Inc., National Cancer Institute at Frederick, Frederick, Maryland Dana L. Vanlandingham Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas Anne K. Vidaver Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, Nebraska Robert A. Wood Department of Pediatrics, Division of Allergy and Immunology, Johns Hopkins University, Baltimore, Maryland Dawn P. Wooley Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, Ohio Abbey K. Woolverton Department of Epidemiology and Biostatistics, Milken Institute School of Public Health, George Washington University, Washington, DC Christopher J. Woolverton Department of Biostatistics, Environmental Health Science and Epidemiology, College of Public Health, Kent State University, Kent, Ohio
- 17. Foreword O n October 29,1997, a non-human primate research worker was transferring macaques from a trans- port cage to a squeeze cage preceding a routine annual physical. One of the macaques became agitated, and as he jumped, his tail flicked material from the bot- tom of the cage into the face and eye of the researcher. On December 10, 1997, that vivacious and talented 22-year- old worker, Elizabeth “Beth” Griffin, died as a result of that innocuous event. Beth’s death was initiated by an ocular exposure to the Herpes simian B virus (Macacine herpesvirus 1). Her case was the first known exposure to be the result of some- thing other than a bite or a scratch. An Agnes Scott Col- lege graduate, Beth—a dancer—died from an encephalitic disease that first paralyzed her from the neck down before finally causing her death. Beth’sdeathgainednationalattentionintheU.S.media. It was a featured story on a network newsmagazine. The incident gained international attention in the world of research. The world—especially the research world— wanted to know how such a thing could ever happen and what could be done to ensure it never happened again. A number of things could have been done that would have meant this story would never be read. There were systematic failures in the occupational health response to her exposure. There were failures in the health care system. There were things Beth could have done, such as wear goggles while handling the monkeys or use the nearby eyewash stations within 5 minutes of her expo- sure. An emergency response measure could have pro- vided a simple postexposure prophylactic prescription taken shortly after her incident. These actions and oth- ers as elements of an institutional culture of safety—Pre- vention, Detection, and Response—could have changed everything. Two years after her death, Beth’s family established a nonprofit foundation to increase safety and occupational health awareness for people who worked with non-hu- man primates. With the collaborative assistance of orga- nizationssuchastheAssociationofPrimateVeterinarians (APV), the American Association for Laboratory Animal Science (AALAS), and the American College of Labora- tory Animal Medicine (ACLAM), many changes were made in processes and responses to exposures. Many people working in non-human primate research environ- ments began carrying cards, quickly tagged as “Beth Cards,” that informed medical personnel to take specific measures to rule out B virus exposure first—not last—if the person was exhibiting certain viral symptoms. In 2003, the world became gripped in an outbreak of a disease called SARS (severe acute respiratory syndrome). The outbreak began in China, but because of mobility the disease soon began popping up elsewhere. As Beth’s death had been a tipping point for safety awareness in working with non-human primates, the SARS outbreak and the global response of expanding laboratory capacity xiii
- 18. to detect andidentify emerging infectious diseases became a massive springboard for biosafety. The "Amerithrax" incident of 2001 had already sparked international attention to practices used in work- ing with certain biological agents. The concepts of bio- safety and biosecurity preceded all of these incidents by decades, but never had there been such total community attention to the potential risks of biological exposures. At the encouragement of those groups with whom we had already collaborated, the Elizabeth Griffin Research Foundation reached out with our “no more Beth Griffin tragedies” message to the American Biological Safety Association to assist in highlighting awareness of and response to the exposure risks that those who work with biological agents face on a rather routine basis. With their assistance—and that of a growing number of similar pro- fessional organizations around the world—biosafety is a front-burner issue in conducting safe and responsible sci- ence. Much has been done to increase the awareness, research, and application of sound protocols that both reduce the risk of exposure and improve the quality of response to an exposure should one occur. The very truth that you are reading this book on biosafety and biosecu- rity is proof enough of how far this has come. Good science is safe science. If the science isn’t safe, it isn’t good. Nothing can be more damaging to the reputa- tion of a research institution or to the public view of the value of science than a bungled exposure issue or the appearanceofcuttingcornersonsafetyinordertoaccom- plish something. Biological risks are very different from many others in that they are most often not immediately evident, due to incubation periods. There are no immedi- ate detection capabilities as with chemical or radiation risks, since biological manifestation may easily be delayed and often misdiagnosed. Compound those issues with the fact that many biological agents have highly contagious, often lethal capabilities, and we quickly see it’s not just the laboratory worker at risk. Watchfulness, attention, caution, and prudence are all required whenever someone does anything that places individuals beyond themselves at risk. To engage in bio- logical research requires that you exercise caution and follow protocols, not only for your safety but also for the safety of the community and world that surrounds you. It is not an option or a luxury. It is a necessity. Every risk, no matter how small it may seem, must be considered, assessed, and properly mitigated. The techniques of safety and security are every bit as important as the tech- niques used in your research. Before getting into the technical nuts and bolts of bio- safety and biosecurity, please keep these basics in mind. 1. Everyone who works with biological agents in any capacity should discuss their work with their personal physician. You are quite possibly the zebra among a stable of horses. 2. Remember that most people drown in shallow water. Whilemuchattentionisrequiredtohigher-riskagents, most laboratory-acquired infections (LAIs) occur when working with what are thought to be lower-risk agents. Most LAI deaths are attributed to Level 2 agents, not Level 3 or 4. 3. Learn from near-misses. Encourage nonpunitive con- versations about things that “almost happened.” The “almost happened” events are likely to recur, so learn from them. 4. Compliance is a by-product of safe research. It is not the purpose of safe research. 5. Be a role model of biosafety and biosecurity. Create atmospheres where being safe appears the most natu- ral thing to do. 6. Link up with the biosafety personnel at your institu- tion. Learn from them. 7. If you think there’s a safer way, don’t just think it. Prove it by research, demonstrate it, and share what you learned with the biosafety community. 8. Commit to never letting a Beth Griffin tragedy happen wherever you may be. We adhere to the words spoken by Thomas Huxley at the opening of The Johns Hopkins University in Baltimore, Maryland. In his remarks, Huxley noted that “the end of life is not knowledge, but action.” On behalf of the Eliza- beth R. Griffin Research Foundation and our collabora- tive partners worldwide, we encourage that you not just learn the material in this book but act upon, promote, and add to this body of knowledge throughout your scientific career. Caryl P. Griffin, MDiv, President and Founder James Welch, Executive Director Elizabeth R. Griffin Foundation www.ergriffinresearch.org xiv | FOREWORD
- 19. Preface I t is witha great sense of honor and reverence that we take over the reins of editing this book from our esteemed colleagues, Diane O. Fleming and Debra L. Hunt. It is our hope that this 5th edition of Biological Safety: Princi ples and Practices remains the main text in the field of biosafety. We are indebted to the many authors who have contributed to this edition. This book serves as a valuable resource not only for biosafety professionals, but also for students, staff, faculty, and clinicians who are working with or around potentially biohazardous materi- als in research laboratories, medical settings, and indus- trial environments. Those who supervise biosafety or laboratory staff members will also benefit from this book. We deci ded to keep the overall structure similar to the previous edition, with five major sections. Eight new chap- ters were added on the following topics: molecular agents, arthropod vector biocontainment, aerobiology, training programs, veterinary and green house biosafety, field stud- ies, and clinical laboratories. Biosafety Practices is not a separate chapter in this edition; the concepts have been incorporated into relevant chapters. Similarly, the infor- mation on prions was incorporated into the new chapter on molecular agents. The title of the last section was changed from “Special Considerations” to “Special Envi- ronments” and some chapters were moved out of this section to keep the focus on unique settings encountered inbiosafetypractice.Sinceregulatoryguidelinesarealways changing, we have directed our readers to online sources for the most up- to- date information. Chapters have been made to be more fluid and stand- alone by minimizing ref- erencestootherchapters.Wearefortunatetohavecolorin this new edition. Both of this edition’s editors are Certified Biosafety Professionals,butwecametothefieldofbiosafetythrough differ ent ave nues, giving us complementary perspectives on the topic. Dawn Wooley became intensely interested in biosafety during her graduate days at Harvard while researching the newly discovered AIDS viruses. These were the days before there were impor tant administrative controls such as the Bloodborne Pathogen Standard. In trying to protect herself and others around her from these newly emerging pathogens, Dawn developed a love for the field of biosafety that has persisted until today. Karen Byers developed a keen interest in biosafety while work- ing with measles in Harvard research laboratories. An appointment to the Institutional Biosafety Committee inspired her to become a biosafety professional. She is very grateful for Lynn Harding’s mentorship and the opportunities for professional development and leader- ship provided by colleagues in the American Biological Safety International (ABSA). Professional organ izations such as ABSA, the American Society of Microbiology (ASM), the American Public Health Association (APHL), the Clinical and Laboratory xv
- 20. xvi | PREFACE Standards Institute (CLSI),and the American Association for Laboratory Animal Science (AALAS) have played a key role in fostering the development and implementa- tion of evidence-based biosafety practice. The Foreword to this edition reminds us of the importance of this endeavor. Gregory W. Payne, Se nior Editor, ASM Press, was instrumental in pushing for the update of this book, and he provided much- needed guidance and inspiration. We thank Ellie Tupper and Lauren Luethy for their expert assistance with the production of this book. We hope that our readers enjoy the book as much as we have appreciated the opportunity to work on it for you and the rest of the biosafety community. Be safe! Dawn P. Wooley Karen B. Byers
- 21. Hazard Identification 1. The Microbiotaof Humans and Microbial Virulence Factors Paul A. Granato | 3 2. Indigenous Zoonotic Agents of Research Animals Lon V. Kendall | 19 3. Biological Safety Considerations for Plant Pathogens and Plant-Associated Microorganisms of Significance to Human Health Anne K. Vidaver, Sue A. Tolin, and Patricia Lambrecht | 39 4. Laboratory-Associated Infections Karen Brandt Byers and A. Lynn Harding | 59 SECTION I 560-65980_sec I_7P.indd 1 2/8/17 9:51 AM
- 22. 560-65980_sec I_7P.indd 22/8/17 9:51 AM
- 23. 3 T he 1990s havebeen marked by a renewed recogni- tion that our human species is still locked in a Darwinian struggle with our microbial and viral predators.” Although this unreferenced quotation was made by Nobel Laureate Joshua Lederberg, as he was dis- cussing the acquired immunodeficiency syndrome (AIDS) and multidrug-resistant Mycobacterium tuberculosis epidemics that emerged in the early 1990s, his comment could also apply to almost any infectious disease process that has occurred since the recognition of the germ theory of disease in the late 1880s. For as we journey through the 21st century, and despite the advances of modern medi- cine and the continual development of new vaccines and anti-infective therapeutic agents, the human species con- tinues to battle microbial predators in this Darwinian struggle for survival. MICROBIOTA ANDTHE HUMAN GENOME PROJECT The human normal flora consists of an ecological com- munity of commensal, symbiotic, and pathogenic micro- organisms in dynamic balance that literally share and inhabit our body spaces throughout life. In 2001, Leder- berg (1) coined the term “microbiota” to describe these microbial communities that were characterized by using cultural methods. Subsequently, in 2008, the Human Mi- crobiome Project (HMP) was funded by the National Institutes of Health to use noncultural methods to study how changes in the human microbiome are associated with health and disease (2). The HMP used genetic- based, molecular methods, such as metagenomics and genome sequencing, to characterize all microbes present in a body site, even those that could not be cultured. As such, by using metagenomics (which provides a broad genetic perspective on a single microbial community) and extensive whole-genome sequencing (which pro- vides a genetic perspective on individual microorganisms in a given microbial community), the HMP provided a more comprehensive understanding of the microorgan- isms that inhabit a particular body site through genetic analysis. The HMP studies (3) have shown that even healthy in- dividuals differ remarkably in the microbes that occupy body sites such as the skin, mouth, intestine, and vagina. “ PAUL A. GRANATO The Microbiota of Humans and Microbial Virulence Factors 1
- 24. 4 | HAZARD IDENTIFICATION Much ofthis diversity remains unexplained, although diet, environment, host genetics, and early microbial exposure have all been implicated. These studies have also led some investigators to conclude that the human microbiome may play a role in autoimmune diseases like diabetes, rheumatoid arthritis, muscular dystrophy, mul- tiple sclerosis, fibromyalgia, and perhaps some cancers (4). Others have proposed that a particular mix of mi- crobes in the intestine may contribute to common obesity (5–7). It has also been shown that some of the microbes in the human body can modify the production of neu- rotransmitters in the brain that may possibly modify schizophrenia, depression, bipolar disorder, and other neurochemical imbalances (8). DYNAMICS OFTHE HOST–PARASITE RELATIONSHIP The dynamics of this host–parasite relationship for sur- vival are in a continual state of change. In health, a bal- ance exists between the host and the microbe that allows for the mutual survival and coexistence of both. This bal- ance is best maintained when humans have operative host defense mechanisms and are not exposed to any par- ticular infectious microbial agent. The three major host defense mechanisms that must be operative to maintain this balance and the health of the human host are (i) in- tact skin and mucous membranes, (ii) a functional group of phagocytic cells consisting principally of the reticulo- endothelial system (RES), and (iii) the ability to produce a humoral immune response. Defects in any one or com- bination or all of these host defense mechanisms will shift the balance in favor of the microbe and predispose the host to the risk of developing an infectious disease process. For example, breaks in skin or mucous mem- branes due to accidents, trauma, surgery, or thermal in- jury may serve as a portal of entry for microorganisms to produce infection. In addition, the inability to phagocy- tize microorganisms effectively by the RES due to lym- phomaorleukemiaandtheinabilitytoproducefunctional humoral antibodies due to defects in plasma cells or ex- posure to immunosuppressive agents (i.e., drugs, irradia- tion, etc.) may also predispose to the development of infection. This balance in favor of the microbe may be shifted back toward the host through the use of antimi- crobial agents and/or the administration of vaccines for the treatment and prevention of disease. Unfortunately, as these agents or selective pressures may adversely af- fect the survival of the microbe, these developments are often followed by a shift in balance back in favor of the ever-adaptable microbe by, perhaps, acquiring new mech- anisms for producing human disease or resisting the action of an antimicrobial agent. The microbial world consists of bacteria, fungi, vi- ruses, and protozoa that represent over several hundred thousand known species. The great majority of these, however, are not involved in any dynamic relationship with the human host because they are incapable of sur- viving or causing disease in humans. By comparison, those microorganisms that are involved in the dynamic relation- ship with the host are limited in number, consisting of fewer than 1,000 known microbial species. It is this limited group of microorganisms that is the focus of discussion in this chapter. The relationships that exist between the human host and the microbial world are varied and complex. When a microorganism that is capable of causing disease be- comes established in the body, this process is called an infection, and an infection that produces symptoms in a human is called an infectious disease. By contrast, persis- tence of microorganisms in a particular body site (such as the normal microbial flora, as is discussed in a subsequent section of this chapter) is often referred to as coloniza- tion rather than infection. Importantly, infection or colo- nization does not necessarily lead to the development of an infectious disease. If host defenses are adequate, a per- son may be infected by a disease-causing microorganism for an indefinite period without any signs or symptoms of disease. Such individuals are referred to as asymptomatic carriers or simply carriers who have asymptomatic or subclinical infection. These asymptomatic carriers serve as important reservoirs for transmission of the infecting organisms to susceptible hosts who may subsequently develop symptomatic disease. The ability of certain microorganisms to infect or cause disease depends on the susceptibility of the host, and there are notable species differences in host suscepti- bility for many infections. For instance, dogs do not get measles and humans do not get distemper. Thus, the term pathogenicity, which is defined as the ability of a micro- organism to cause disease, must be qualified according to the host species involved. Microorganisms that do not normally produce disease in the healthy human host are often called saprophytes, commensals, or nonpathogens. In recent years, increasing numbers of infectious dis- eases have been caused by microorganisms that were previously considered nonpathogenic. These infectious diseases often develop in patients whose surface/barrier, cellular, or immunologic defenses are compromised by such things as trauma, genetic defects, underlying dis- ease, or immunosuppressive therapy. Microorganisms that are frequent causes of disease only in the immuno- compromised host or when skin or mucosal surfaces or barriers are breached are called opportunistic pathogens. Opportunistic pathogens are often saprophytes that rarely cause disease in individuals with functional host defense mechanisms.
- 25. CHAPTER 1: THEMICROBIOTA OF HUMANS AND MICROBIAL VIRULENCE FACTORS | 5 Pathogenicity refers to the ability of a microorganism to cause disease, and virulence provides a quantitative measure of this property. Virulence factors refer to the properties that enable a microorganism to establish itself on or within a host and enhance the organism’s ability to produce disease. Virulence is not generally attributable to a single discrete factor but depends on several para meters related to the organism, the host, and their inter- action. Virulence encompasses two general features of a pathogenic microorganism: (i) invasiveness, or the ability to attach, multiply, and spread in tissues, and (ii) toxige- nicity, the ability to produce substances that are injurious to human cells. Highly virulent, moderately virulent, and avirulent strains may occur within a single species of organisms. The microorganisms that cause human infectious dis- eases are acquired from two major sources or reservoirs: those acquired from outside the body, called exogenous reservoirs, and those infectious diseases that result from microorganisms that inhabit certain body sites, called endogenous reservoirs. Most exogenous infections are acquired from other individuals by direct contact, by aero- sol transmission of infectious respiratory secretions, by ingestion of contaminated food or drink, or indirectly through contact with contaminated inanimate objects (often called fomites). Some exogenous infections may also be acquired by puncture of the skin during an insect or animal bite and, perhaps, by occupational exposure from sharps. Endogenous infections occur more com- monly than exogenous infections and are acquired from microorganisms that reside normally on various body sites (called normal commensal flora) gaining access to anatomic sites that are normally sterile in health. NORMAL MICROBIAL FLORA The terms “normal microbial flora,” “normal commensal flora,” “indigenous flora,” and “microbiota” are often used synonymously to describe microorganisms that are fre- quently found in particular anatomic sites in healthy in- dividuals, whereas the term “microbiome” refers to their genomes. This microbial flora is associated with the skin and mucous membranes of every human from shortly af- ter birth until death and represents an extremely large and diverse population of microorganisms. The healthy adult consists of about 10 trillion cells and routinely har- bors at least 100 trillion microbes (9). The entire microbi- ome accounts for about 1% to 3% of the total human body mass (10) with some weight estimates ranging as high as 3 pounds or 1,400 grams. The constituents and numbers of the flora vary in different anatomic sites and some- times at different ages. They comprise microorganisms whose morphologic, physiologic, and genetic properties allow them to colonize and multiply under the conditions that exist in a particular body site, to coexist with other colonizing organisms, and to inhibit competing intrud- ers. Thus, each anatomic site that harbors a normal mi- crobial flora presents a particular environmental niche for the development of a unique microbial ecosystem. Local physiologic and environmental conditions at various body sites determine the nature and composition of the normal flora that exists there. These conditions are sometimes highly complex, differing from site to site, and sometimes vary with age. Some of these local anatomic conditions include the amounts and types of nutrients available for microbial growth, pH, oxidation reduction potentials, and resistance to local antibacterial substances, such as bile, lysozyme, or short-chain fatty acids. In addi- tion, many bacteria have a remarkable affinity for specific types of epithelial cells to which they adhere and on which they multiply. This adherence, which is mediated by the presence of bacterial pili/fimbriae or other micro- bial surface components, allows the microbe to attach to specific receptor sites found on the surface of certain epithelial cells. Through this mechanism of adherence, microorganisms are permitted to grow and multiply while avoiding removal by the flushing effects of surface fluids and peristalsis. Various microbial interactions also determine their relative prevalence in the flora. Some of these interactions include competition for nutrients and inhibition of growth by the metabolic products pro- duced by other microorganisms in the ecosystem (for ex- ample, the production of hydrogen peroxide, antibiotics, and/or bacteriocins). The normal microbial flora plays an important role in health and disease. In health, for example, the normal microbial flora of the intestine participates in human nutrition and metabolism. Certain intestinal bacteria syn- thesize and secrete vitamin K, which can then be absorbed by the bowel for use in the human. In addition, the metab- olism of several key compounds involves excretion from the liver into the intestine and their return from there to the liver. This enterohepatic circulatory loop is particu- larly important for the metabolism of steroids and bile salts. These substances are excreted through the bile in conjugated form as glucuronides or sulfates but cannot be reabsorbed in this form. Certain members of the bac- terial intestinal flora make glucuronidases and sulfatases that can deconjugate these compounds, thereby allowing their reabsorption and use by the human host (11, 12). Another beneficial role of the normal microbial flora is the antigenic stimulation of the host’s immune system. Although the various classes of the immunoglobulins pro- duced from this antigenic exposure are usually present in low concentrations, their presence plays an important role in host defense. In particular, various classes of the immunoglobulin A (IgA) group of antibodies produced
- 26. 6 | HAZARD IDENTIFICATION in responseto this antigenic stimulation are secreted through mucous membranes. The role of these immuno- globulins is not well understood, but they may contribute to host defense by interfering with the colonization of deeper tissues by certain normal flora organisms. Perhaps one of the most important roles of the normal microbial flora is to help prevent infectious disease fol- lowing exposure to potential microbial pathogens. The normal commensal flora has the physical advantage of previous occupancy on skin and mucous membranes. Many of these commensal microorganisms adhere to epi- thelial binding sites, thereby preventing attachment to that receptor site by a potential microbial pathogen. As is discussed later in this chapter, certain pathogens that are incapable of adhering to their specific epithelial recep- tors are incapable of causing human disease. In addition, some commensal microorganisms are capable of produc- ing antibiotics, bacteriocins, or other products that may be inhibitory or lethal to pathogenic microorganisms. The collective effect of the normal flora’s ability to adhere to epithelial receptor sites and to produce antimicrobial sub- stances plays an important role in maintaining the health of the host following exposure to a potential microbial pathogen. The normal microbial flora, although important for the maintenance of human health, is a critical factor in human infectious disease. Because the human body is col- onized with diverse and large populations of microorgan- isms as part of one’s normal flora, the three major host defense mechanisms (intact mechanical surfaces, RES, and immune system) must be continually operative and functional for the maintenance of human health in this continually dynamic relationship between the host and parasite. On occasion, normal flora organisms may gain entry into normally sterile body sites, or defects in one or more of the host’s defense mechanisms may result in the development of symptomatic infection from one or more of these organisms. These endogenous human infections occur more fre- quently than those that are acquired from an exogenous source. In general, physicians see more patients with in- fectious diseases acquired from one’s normal microbial flora than those infectious disease processes that are ac- quired from outside the body (13). It is for these reasons that clinicians and clinical microbiologists must be know ledgeable as to the various microbes that reside as the normal flora in different anatomic sites. In medicine, it is often said, “Common things occur commonly.” Knowing the normal microbial flora at a par- ticular anatomic site is often useful in predicting the likely etiologic agents of infection when a neighboring tissue becomes infected from an endogenous source. Therefore, the normal microbial flora for various anatomic sites is reviewed in the following section. Because the residents of the normal microbial flora may vary with the age of the host, this discussion also addresses the normal flora typically found in both healthy newborns and adults when differences in microbial ecosystems may exist. Skin Human skin is a complex microbial ecosystem. The heal thy fetus is sterile in utero until the birth membranes rupture. During and after birth, the infant’s skin is ex- posed to the mother’s genital tract flora, to skin flora from the mother and other individuals who handle the baby, and to a variety of microorganisms acquired by direct con- tact of the baby with the environment. During the infant’s first few days of life, the nature of its microbial skin flora often reflects chance exposure to microorganisms that can grow on particular sites in the absence of microbial competitors. Subsequently, as the infant is exposed to a full range of human environmental organisms, those best adapted to survive on particular skin sites predominate and establish themselves as part of the resident skin flora. Thereafter, the normal microbial flora resembles that of adult individuals. The pH of the skin is usually about 5.6. This factor alone may be responsible for inhibiting the establishment of many microbial species. Despite this, skin provides excellent examples of various microenvironments. Some areas are moist, such as the toe webs and perineum, whereas some areas are relatively dry, such as the fore- arm. Sebaceous glands found on the face, scalp, and upper chest and back produce an abundance of lipids on the skin, whereas other areas, such as the axillae, produce specialized secretions from apocrine glands. Eccrine glands, also called merocrine glands or simply sweat glands, are found in the skin of virtually all ana- tomic sites of the body. These glands produce a clear, odor- less secretion consisting primarily of water and saline that is induced following exposure to high temperature or exercise. As a result of these differences in microen- vironments, quantitative differences in microbial flora occur in each of the three major regions of skin: (i) axilla, perineum, and toe webs; (ii) hands, face, and trunk; and (iii) arms and legs (14). These quantitative differences are the result of differences in skin surface temperature and moisture content as well as the presence of different con- centrations of skin surface lipids that may be inhibitory or lethal to various groups of microorganisms at each of these skin sites (15). The major groups of microorganisms that are normal residents of skin, even though their numbers may vary as influenced by the microenvironment, include various genera of bacteria and the lipophilic yeasts of the genus Malassezia. Nonlipophilic yeasts, such as Candida spe- cies, are also inhabitants of the skin (14). Other bacterial
- 27. CHAPTER 1: THEMICROBIOTA OF HUMANS AND MICROBIAL VIRULENCE FACTORS | 7 species may be found less commonly on the skin, and some of these include hemolytic streptococci (especially in children), atypical mycobacteria, and Bacillus species. The predominant bacterial inhabitants of the skin are the coagulase-negative staphylococci, micrococci, sap- rophytic Corynebacterium species (diphtheroids), and Propionibacterium species. Among this group, Propionibac terium acnes is the best studied because of its association with acne vulgaris. P. acnes is found briefly on the skin of neonates, but true colonization begins during the 1 to 3 years prior to sexual maturity, when numbers rise from less than 10 CFU/cm2 to about 106 CFU/cm2 , chiefly on the face and upper thorax (16). Various species of coagu- lase-negative staphylococci are found as normal inhabit- ants of skin, and some of these include Staphylococcus epidermidis,S.capitis,S.warneri,S.hominis,S.haemolyticus, S. lugdunensis, and S. auricularis (17–20). Some of these staphylococci demonstrate ecological niche preferences at certain anatomic sites. For example, S. capitis and S. au ricularis show an anatomic preference for the head and the external auditory meatus, respectively, whereas S. hominis and S. haemolyticus are found principally in areas where there are numerous apocrine glands, such as the axillae and pubic areas (17). Staphylococcus aureus regu- larly inhabits the external nares of about 30% of healthy individuals and the perineum, axillae, and toe webs of about 15%, 5%, and 2%, respectively, of healthy people (14). Micrococcus spp., particularly Micrococcus luteus, are also found on the skin, especially in women and chil- dren, where they may be present in large numbers. Aci netobacter spp. are found on the skin of about 25% of the population in the axillae, toe webs, groin, and antecubital fossae. Other Gram-negative bacilli are found more rarely on the skin, and these include Proteus and Pseudomonas in the toe webs and Enterobacter and Klebsiella on the hands. Saprophytic mycobacteria may occasionally be found on the skin of the external auditory canal and of the genital and axillary regions, whereas hemolytic strep- tococci tend to colonize the skin of children but not adults (14). The principal fungal flora is Malassezia, a yeast. Der- matophytic fungi may also be recovered from the skin in the absence of disease, but it is unclear whether they rep- resent the normal flora or transient colonizers. Carriage of Malassezia spp. probably reaches 100% in adults, but proper determination of carriage rates is obscured by the difficultyofgrowingsomespeciesoftheselipophilicyeasts in the laboratory (14). Members of the skin microflora live both on the skin surface in the form of microcolonies and in the ducts of hair follicles and sebaceous glands (14). Wolff et al. (21) proposed that Malassezia species live near the opening of the duct, the staphylococci further down, and the pro- pionibacteria near the sebaceous glands. A more recent study (22), however, suggests that all three microbial groups are more evenly distributed throughout the fol- licles. In any event, organisms in the follicles are se- creted onto the skin surface along with the sebum, but staphylococci, at least, also exist in microcolonies on the surface. These microcolonies may be of various sizes and are larger (103 to 104 cells per microcolony) on areas such as the face than on the arms (101 to 102 cells per microcolony) (14). Washing may decrease microbial skin counts by 90%, but normal numbers are reestablished within 8 h (23). Abstinence from washing does not lead to an increase in numbers of bacteria on the skin. Normally, 103 to 104 organisms are found per square centimeter. However, counts may increase to 106 /cm2 in more humid areas, such as the groin and axilla. Small numbers of bacteria are dispersed from the skin to the environment, but cer- tain individuals may shed up to 106 organisms in 30 min of exercise. Many of the fatty acids found on the skin may be bacterial products that inhibit colonization by other species. The flora of hair is similar to that of the skin (24). Eye The normal microbial flora of the eye contains many of the bacteria found on the skin. However, the mechanical action of eyelids and the washing effect of the eye secre- tions that contain the bacteriolytic enzyme lysozyme serve to limit the populations of microorganisms normally found on the eye. The predominant normal microbial flora of the eye consists of coagulase-negative staphylococci, diphtheroids, and, less commonly, saprophytic Neisseria species and viridans group streptococci. Ear The microbiota of the external ear is similar to that of skin, with coagulase-negative staphylococci and Coryne bacterium species predominating. Less frequently found are Bacillus, Micrococcus, and saprophytic species of Neis seria and mycobacteria. Normal flora fungi include Asper gillus, Alternaria, Penicillium, and Candida. RespiratoryTract Nares In the course of normal breathing, many kinds of microbes are inhaled through the nares to reach the upper respira- tory tract. Among these are aerosolized normal soil in- habitantsaswellaspathogenicandpotentiallypathogenic bacteria, fungi, and viruses. Some of these microorgan- isms are filtered out by the hairs in the nose, whereas others may land on moist surfaces of the nasal passages, where they may be subsequently expelled by sneezing or
- 28. 8 | HAZARD IDENTIFICATION blowing one’snose. Generally, in health these airborne microorganisms are transient colonizers of the nose and do not establish themselves as part of the resident com- mensal flora. The external 1 cm of the external nares is lined with squamous epithelium and has a flora similar to that found on the skin, except that S. aureus is commonly carried as the principal part of the normal flora in some individuals. Approximately 25% to 30% of healthy adults in the com- munity harbor this organism in their anterior nares at any given time, 15% permanently and the remaining 15% transiently (25). Nasopharynx Colonization of the nasopharynx occurs soon after birth following aerosol exposure of microorganisms from the respiratory tract from those individuals who are in close contact with the infant (i.e., the mother, other family members, etc.). The normal microbial flora of the infant establishes itself within several months and generally remains unchanged throughout life. The nasopharynx has a flora similar to that of the mouth (see below) and is the site of carriage of potentially pathogenic bacteria such as Neisseria meningitidis, Branhamella catarrhalis, Streptococcus pneumoniae, S. aureus, and Haemophilus influenzae (25). The respiratory tract below the level of the larynx is protected in health by the actions of the epiglottis and the peristaltic movement of the ciliary blanket of the colum- nar epithelium. Thus, only transiently inhaled organisms are encountered in the trachea and larger bronchi. The accessory sinuses are normally sterile and are protected in a similar fashion, as is the middle ear, by the epithelium of the eustachian tubes. GastrointestinalTract Mouth Colonization of the mouth begins immediately following birth when the infant is exposed to the microorganisms in the environment, and the numbers present increase rapidly in the first 6 to 10 h after birth (26). During the first few days, several species appear sporadically as tran- sients, many of them not being suitable for the oral envi- ronment. During this period, the oral mucosa becomes colonized by its first permanent residents; these are de- rived mainly from the mouth of the mother and other persons in contact with the infant (26, 27). The child is continuously exposed to transmission of oral bacteria from family members by direct and indirect contact (the latter, for example, via spoons and feeding bottles), as well as by airborne transmission. The various members of the resi- dent microflora become established gradually during the first years of life as growth conditions become suitable for them. This microbial succession is caused by environ- mental changes related to the host, such as tooth eruption or dietary changes, as well as to microbial interrelations due to, for example, the initial colonizers reducing tissue redox potentials or supplying growth factors. During the first months of life, the oral microflora mainly inhabits the tongue and is dominated by strep- tococci, with small numbers of other genera such as Neis seria, Veillonella, Lactobacillus, and Candida. Streptococcus salivarius is regularly isolated from the baby’s mouth starting from the first day of life, and often the bacteriocin types are identical to those of the mother (28). Strepto coccus sanguinis colonizes the teeth soon after eruption (29), whereas Streptococcus mutans colonizes much more slowly over several years, starting in pits and fissures and spreading to proximal and other surfaces of the teeth (30). Colonization with S. mutans and lactobacilli is correlated with dental caries (29, 31), and, in fact, their establishment can be inhibited or delayed by caries-preventive measures in the infants’ mothers (32). Dental caries result from the ability of these bacteria to produce biofilms that adhere to the tooth surface. Biofilms and their relationship to microbial virulence will be discussed later in the Viru- lence Factors and Mechanisms section of this chapter. As dental plaque forms on the erupting teeth, the oral microflora becomes more complex and predominately anaerobic. Studies of 4- to 7-year-olds have shown the plaque microflora in the gingival area to be similar to that in adults, with motile rods and spirochetes observed by direct microscopy, and the same species of Actinomyces, Bacteroides, Capnocytophaga, Eikenella, etc., recovered by cultural techniques (33–36). In studies of 7- to 19-year- olds, the prevalence of some organisms and the propor- tions they constitute of the flora seem, however, to differ with age and hormonal status. Thus, Prevotella species and spirochetes increase around puberty, while Actino myces naeslundii and Capnocytophaga spp. tend to de- crease with increasing age of the children. In healthy adults, the resident oral microflora consists of more than 200 Gram-positive and Gram-negative bacterial species as well as several different species of mycoplasmas, yeasts, and protozoa. Only about 100 oral species of bacte- riahaveknowngenusspeciesnamesbaseduponbiochemi cal and physiologic characteristics (37). With the eruption of teeth and the development of gingival crevices, anaer- obic bacteria emerge as the principal flora of the mouth. Concentrations of bacteria vary from approximately 108 CFU/ml in the saliva to 1012 CFU/ml in the gingival crevices around teeth, with the anaerobic bacteria outnumbering the aerobic bacteria by a ratio of a least 100:1. The mouth has several different habitats where micro- organisms can grow. Each habitat has its own unique envi- ronment and is populated by a characteristic community of microorganisms consisting of different populations of
- 29. CHAPTER 1: THEMICROBIOTA OF HUMANS AND MICROBIAL VIRULENCE FACTORS | 9 various species in each ecosystem. Each species performs a certain functional role as part of the microbial commu- nity. Some of the major ecosystems may be found on mucosal surfaces of the palate, gingiva, lips, cheeks, and floor of the mouth, the papillary surface of the tongue, andtoothsurfaces,withtheirassociateddentalplaque,gin- gival pockets, etc. To remain in the mouth, the micro- organisms must adhere to the oral surfaces, resist being eliminated with the stream of saliva swallowed, and grow under the different conditions prevailing at each site. Such sites can harbor extremely numerous and complex mi- crobial communities. For detailed and comprehensive information, the reader is referred to the review by Theilade (37). In general, streptococcal species constitute 30% to 60% of the bacterial flora of the surfaces within the mouth. These are primarily viridans group streptococci: S. sali varius, S. mutans, S. sanguinis, and S. mitis, found on the teeth and in dental plaque. Specific binding to mucosal cells or to tooth enamel has been demonstrated with these organisms. Bacterial plaque developing on the teeth may contain as many as 1011 streptococci per gram in addition to actinomycetes and Veillonella and Bacteroides species. Anaerobic organisms, such as Prevotella melaninogenica, treponemes, fusobacteria, clostridia, propionibacteria, and peptostreptococci, are present in gingival crevices, where the oxygen concentration is less than 0.5%. Many of these organisms are obligate anaerobes and do not survive in higher oxygen concentrations. The natural habitat of the pathogenic species Actinomyces israelii is the gingival crevice. Among the fungi, species of Candida and Geot richum are found in 10% to 15% of individuals (37). Esophagus Little attention has been given to characterizing the nor- mal microflora of the esophagus. Essentially, the esopha- gus is a transit route for food passing from the mouth to the stomach, with approximately 1.5 liters of saliva swal- lowed per day (38, 39). Although much of this stimulated saliva is swallowed with food, there is a resting rate of sa- liva secretion estimated to be about 20 ml/h (38), and this saliva is swallowed as fluid. In addition, nasal secretions containing the microbial flora of that site may also be swallowed, introducing salt-tolerant organisms, such as staphylococci, from the anterior and posterior nares. Consequently, normal flora mouth and nasal micro organisms will be recovered from the esophagus, but it is uncertain whether these organisms represent tran- sient colonization or an established microflora. Stomach As for the esophagus, oral and nasal normal flora microor- ganisms, as well as microorganisms ingested in food and drink, are swallowed into the stomach. However, the vast majority is destroyed following exposure to the gastric acid (pH 1.8 to 2.5) (40). Concentrations of bacteria in the healthy stomach are generally low, less than 103 CFU/ml, and are composed primarily of relatively acid-resistant species, such as gastric helicobacters, streptococci, staph- ylococci, lactobacilli, fungi, and even smaller numbers of peptostreptococci, fusobacteria, and Bacteroides spe- cies (41–43). Gram-positive organisms predominate in the stomach, with a striking absence of Enterobacteriaceae as well as Bacteroides and Clostridium species. The gastric flora can become more complex when the ability to achieve an acid pH is altered by the buffering action of food, by hypochlorhydria due to an intrinsic pathogenic process or surgery (40), or by the medicinal use of proton pump inhibitors, such as omeprazole. In the newborn, the stomach secretes very little gastric acid and does not achieve optimal acid secretion rates until 15 to 20 days after birth (41). Consequently, during the first few days of life, the stomach does not constitute a microbicidal barrier to gut colonization. Intestine A fecal flora is acquired soon after birth (44). The compo- sition of the early flora depends on a number of factors, including the method of delivery, the gestational age of the newborn infant, and whether the infant is breast- or bottle-fed. After vaginal delivery, the newborn gut is first colo- nized by facultative organisms acquired from the moth- er’s vaginal flora, mainly Escherichia coli and streptococci (44). The guts of infants delivered by cesarean section are usually colonized by Enterobacteriaceae other than E. coli with a composition resembling the environmental flora of the delivery room (45). Anaerobes appear within the first week or two of life and are acquired more uniformly and more rapidly in bottle-fed than in breast-fed babies. Virtually 100% of full-term, bottle-fed, vaginally deliv- ered infants have an anaerobic flora within the first week of life, with Bacteroides fragilis predominating, whereas only 59% of similarly delivered but breast-fed infants have anaerobes at this time, and less than 10% harbor B. fragilis (46). Breast-fed infants have a marked predomi- nance of Bifidobacterium spp. in their colons that exceed the number of Enterobacteriaceae 100- to 1,000-fold (47). The nature of the gut flora may be influenced by the nutrient content of breast or cow’s milk, compared to that of infant formulas that are fortified with nutrients such as iron. The presence of iron seems to stimulate a complex flora composed of Enterobacteriaceae, Clostrid ium species, and Bacteroides species. The low-iron breast or cow’s milk diet selects for a simple flora composed predominately of Bifidobacterium species and Lactoba cillus species (48, 49). In breast-fed infants, the Bifidobac terium population increases in the first few weeks of life
- 30. 10 | HAZARD IDENTIFICATION to becomethe stable and dominant component of the fecal flora until the weaning period (50, 51). The properties of breast milk that promote the dominance of Gram-positive bacilli in the feces are not known with certainty but no doubt involve both nutritional and immunologic factors. Weaning produces significant changes in the composi- tion of the gut flora resulting in increased numbers of E. coli, Streptococcus, Clostridium, Bacteroides, and Pepto streptococcus species. After weaning, a more stable adult- type flora occurs, in which the number of Bacteroides organisms equals or exceeds the number of Bifidobac terium organisms, with E. coli and Clostridium counts decreasing (16). In adults, the composition of the fecal flora appears to vary more from individual to individual than it does in particular subjects studied over time (41, 43, 52). Bacteria make up most of the flora in the colon and account for up to 60% of the dry mass of feces (53). From 300 (54) to 1,000 (55) different bacterial species reside in the gut, with most estimates at about 500 (56–58). However, it is likely that 99% of the intestinal bacteria are repre- sented by 30 to 40 species (59). Fungi and protozoa also make up part of the gut flora, but little is known about their activities. The numbers and types of bacteria found in the small intestine depend on the flow rate of intestinal contents. When stasis occurs, the small intestine may contain an extensive, complex microbial flora. Normally, flow is brisk enough to wash the microbial flora through to the distal ileum and colon before the microorganisms multi- ply. Consequently, the types and numbers of microbes encountered in the duodenum, the jejunum, and the ini- tial portions of ileum are similar to those found in the stomach and on average comprise 103 CFU/ml (60–63). Anaerobes only slightly outnumber facultative organisms, with streptococci, lactobacilli, yeasts, and staphylococci also found. As the ileocecal valve is approached, the number and variety of Gram-negative bacteria begin to increase (34, 42, 64). Coliforms are found consistently, and the numbers of both Gram-positive and Gram-negative anaerobic organisms (such as Bifidobacterium, Clostridium, Bacteroi des, and Fusobacterium) rise sharply to 105 to 106 CFU/ml on average. In the adult colon, another dramatic increase in the microbial flora occurs as soon as the ileocecal valve is crossed. Here, the number of microorganisms present approaches the theoretical limits of packing cells in space. Nearly one-third of the dry weight of feces consists of bacteria, with each gram of stool containing up to 1011 to 1012 organisms (65). This microbial number is about 1 1og greater than the total number of cells in the entire human body (66, 67). Over 98% of the organisms found in the colon are strict anaerobes, with the anaerobes outnumbering aerobes 1,000- to 10,000-fold. The distribution of the major genera of organisms found in the colon per gram of feces is as follows: Bacteroides, 1010 to 1011 ; Bifidobacterium, 1010 to 1011 ; Eubacterium, 1010 ; Lactobacillus, 107 to 108 ; coli- forms, 106 to 108 ; aerobic and anaerobic streptococci, 107 to 108 ; Clostridium, 106 ; and yeasts at variable numbers (24). Thus, more than 90% of the fecal flora consists of Bacteroides and Bifidobacterium. Intensive studies of the colonic microbial flora have shown that the average healthy adult harbors well over 200 given species of bacteria alone. Benefits of intestinal flora The intestinal microbiota performs many important func- tions for the host to maintain health and life. Without gut flora, the human body would not be able to utilize some of the undigested carbohydrates consumed because some gut flora possess enzymes that human cells lack for hy- drolyzing certain polysaccharides (55). In addition, bac- teria can ferment carbohydrates to produce acetic acid, propionic acid, and butyric acid that can be used by host cells to provide a major source of useful energy and nutri- ents (58, 59). Intestinal bacteria can also assist in absorb- ing dietary minerals such as calcium, magnesium, and iron (54). Gut bacteria can enhance the absorption and stor- age of lipids (55) and produce essential vitamins, such as vitamin K, that are subsequently absorbed by the intestine for use by the human host. The normal gut microbiota plays a role in defense against infection by preventing harmful bacterial spe- cies from colonizing the gut through competitive ex- clusion, an activity often referred to as the “barrier effect.” Harmful bacterial species, such as Clostridium difficile, the overgrowth of which can cause pseudo- membranous colitis, are unable to grow excessively due to competition from helpful gut flora. These microor- ganisms adhere to the mucosal lining of the intestine, thereby pre venting the attachment and potential over- growth of potentially pathogenic species (54). Gut flora also play important roles in establishing the host’s sys- temic immunity (54, 56, 57), preventing allergies (68), and preventing inflammatory bowel disease, such as Crohn’s disease (69). GenitourinaryTract Urethra The only portion of the urinary tract in both males and females that harbors a normal microbial flora is the distal 1 to 2 cm of the urethra. The remainder of the urinary tract is sterile in health. The microbial flora of the distal portion of the urethra consists of various members of the Enterobacteriaceae, with E. coli predominating. Lactoba- cilli, diphtheroids, alpha-hemolytic and nonhemolytic
- 31. CHAPTER 1: THEMICROBIOTA OF HUMANS AND MICROBIAL VIRULENCE FACTORS | 11 streptococci, enterococci, coagulase-negative staphylo- cocci, Peptostreptococcus species, and Bacteroides species are also found. In addition, Mycoplasma hominis, Urea plasma urealyticum, Mycobacterium smegmatis, and Can dida species may also be recovered from this anatomic site in health (25). Vagina The normal microbial flora of the vagina varies according to hormonal influences at different ages (70). At birth, the vulva of a newborn is sterile, but after the first 24 h of life, it gradually acquires a rich and varied flora of saprophytic organisms, such as diphtheroids, micrococci, and non- hemolytic streptococci. After 2 to 3 days, estrogen from the maternal circulation induces the deposition of glyco- gen in the vaginal epithelium, which favors the growth of lactobacilli. The lactobacilli produce acid from glyco- gen that lowers the pH of the vagina, and a resultant mi- crobial flora develops that resembles that in a pubertous female. The low pH created by the lactic acid produced by lac- tobacilli serves as an important host defense mechanism in puberty by preventing the growth of potential vaginal pathogens such as Gardnerella vaginalis, Mobiluncus spp., Neisseria gonorrhoeae, and S. aureus (71–74). In addition, lactobacilli help to prevent colonization of potentially pathogenic microorganisms by avidly adhering to recep- tor sites on the vaginal epithelium, thereby preventing attachment of pathogenic microorganisms and reducing the possibility of infection (75). In addition, up to 98% of lactobacilli may also produce hydrogen peroxide, which has been shown to inactivate human immunodeficiency virus type 1 (HIV-1), herpes simplex virus 2, Trichomonas vaginalis, G. vaginalis, and E. coli (76, 77). Collectively, the production of lactic acid and hydrogen peroxide by lacto- bacilli serves as important host defense mechanisms in preventing many vaginal infections. After the passively transferred estrogen is excreted, the glycogen disappears, with the resultant loss of lacto- bacilli as the predominant vaginal flora and the increase of pH to a physiologic or slightly alkaline level. At this time, the normal microbial flora is mixed, nonspecific, and relatively scanty and contains organisms derived from the floras of the skin and colon. At puberty, the gly- cogen reappears in the vaginal epithelium and the adult microbial flora is established. The predominant flora of the vagina in puberty consists of anaerobic bacteria in concentrations of 107 to 109 CFU/ml of vaginal secretion; these outnumber the aerobic bacteria 100-fold. The major groups of microorganisms represented include lactoba- cilli, diphtheroids, micrococci, coagulase-negative staph- ylococci, Enterococcus faecalis, microaerophilic and anaerobic streptococci, mycoplasmas, ureaplasmas, and yeasts. During pregnancy, the anaerobic microflora de- creases significantly, whereas the numbers of aerobic lactobacilli increase 10-fold (78, 79). The vaginal flora in postmenopausal women is poorly studied. Specimens are often difficult to obtain from healthy women in this category because they seldom pres- ent to a physician unless with some gynecological prob- lem and because the amount of vaginal secretion produced and available for sampling is greatly reduced. However, at least one report (80) documents a significant decrease in lactobacilli in the vaginal flora in postmenopausal women due to the lack of circulating estrogen and the resultant decrease in glycogen in the vaginal mucosa. VIRULENCE FACTORS AND MECHANISMS The factors that determine the initiation, development, and outcome of an infection involve a series of complex and shifting interactions between the host and the para- site, which can vary with different infecting microorgan- isms. In general, humans are able to resist infection by having functional host defense mechanisms. On occa- sion, defects in host defense mechanisms or exposure to a particularly virulent microbial agent may predispose to the development of an infectious disease. The microbial factors that contribute to the virulence of a microorgan- ism can be divided into three major categories: (i) those that promote colonization of host surfaces, (ii) those that evade the host’s immune system and promote tissue inva- sion, and (iii) those that produce toxins that result in tissue damage in the human host. Pathogenic micro organisms may have any, or all, of these factors. Colonization Factors Adherence Most infections are initiated by the attachment or adher- ence of the microbe to host tissue, followed by microbial replication to establish colonization. This attachment can be relatively nonspecific or can require the interaction between structures on the microbial surfaces and specific receptors on host cells. This adherence phenomenon is particularly important in the mouth, small intestine, and urinary bladder, where mucosal surfaces are washed continually by fluids. In these areas, only microorgan- isms that can adhere to the mucosal surface can colo- nize that site. Bacteria adhere to tissues by having pili and/or adhes- ins. Pili or fimbriae are rod-shaped structures that consist primarily of an ordered array of a single protein subunit called pilin. The tip of the pilus mediates adherence of bacteria by attaching to a receptor molecule on the host cell surface that is composed of carbohydrate residues of either glycoproteins or glycolipids. The binding of the
- 32. 12 | HAZARD IDENTIFICATION pilus toits host target cell can be quite specific and ac- counts for the tissue tropism associated with certain bac- terial infections. Bacterial pili are easily broken and lost and have to be continually regenerated by the bacterium. An important function of pilus replacement, at least for some bacteria, is that it provides a way for the bacterium to evade the host’s immune response. Host antibodies that bind to the tips of pili physically block the pili from binding to their host cell targets. Some bacteria can evade this immune defense by growing pili of different anti- genic types, thereby rendering the host’s immune re- sponse ineffective. For example, N. gonorrhoeae can pro- duce over 50 pilin types that make it virtually impossible for the host to mount an antibody response that prevents colonization (81). Bacterial adherence can also be accomplished by a process involving bacterial cell-surface structures known as adhesins and complementary receptors on the surface of host cells. These adhesins, also known as afimbrial adhesins, are proteins that promote the tighter binding of bacteria to host cells following initial binding by pili. The mechanisms used by a microorganism to adhere to a host cell dictate its ability to enter the cell and set in motion a number of physiologic events. An elegant example of mi- crobial attachment followed by a sequence of pathologi- cal effects is that of enteropathogenic E. coli. Following initial adhesion, intracellular calcium levels increase, activating actin-severing enzymes and protein kinases, which then lead to vesiculation and disruption of the microvilli. The bacteria are then able to attach to the epi- thelium in a more intimate fashion, allowing maximal activation of protein kinases. This results in major changes to the cytoskeleton and alterations in the perme- ability of the membrane to ions. Changes in ion perme- ation result in ion secretion and reduction in absorption, resulting in the secretory diarrhea that is the hallmark of this disease. It has been found that a majority of entero- pathogenic E. coli isolates contain a large plasmid that codes for its adhesive properties (82). Biofilms Microbial biofilms develop when microorganisms adhere irreversibly to a submerged surface and produce extra- cellular polymers that facilitate adhesion and provide a structural matrix. The surface may be living tissue, such as teeth or mucosal cells, or inert, nonliving material, such as indwelling medical devices that have been in- serted into the body. Most biofilms are caused by bacte- ria, but they can also be caused by fungi, particularly yeast. These biofilms are complex aggregates of extracel- lular polymers produced by the microorganism growing on a solid animate or inanimate surface that are charac- terized by a chemical heterogenicity and structural diver- sity. On human tissue, the first or basal layer of bacteria or yeast attaches directly to the surface of the host cells and other layers of the microorganism are attached to the basal layer by a polysaccharide matrix. Biofilms have been detected in the vagina, mouth, and intestine, and, in fact, the resident microfloras of these sites may largely be organized into biofilms. These dense mats of organisms may help explain the barrier function of these sites in protection of the host. However, the formation of bio- films may also be the prelude to disease. For example, dental plaque is a biofilm that is known to cause disease, such as caries and gingivitis, and Pseudomonas aerugi nosa has been shown to establish pathogenic biofilms in the lungs of cystic fibrosis patients. Biofilms may also form on foreign objects that have been implanted in the human host or come in repeated contact with human tissue. Biofilms can develop on virtu- ally any indwelling medical device, such as central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pace- makers, prosthetic joints, and urinary catheters. Indeed, hospital-acquired infections in patients with such in- dwelling medical devices are generally preceded by the formation of a biofilm on the surface of the foreign object. Microorganisms within biofilms are imbedded within the extracellular polymer matrix, which makes them highly resistant to antibiotic treatment. For this reason, individuals with such infections invariably require surgi- cal replacement of the prosthesis or removal of the catheter or central line because these infections are re- fractory to antimicrobial therapy. Biofilm formation on embedded plastic and stainless steel devices provides yet another example of well-intentioned iatrogenic activities that continue to create new niches for microorganisms to exploit as causes of human infection. Iron acquisition mechanisms Once a microorganism adheres to a body site, it has an obligate requirement for iron for its subsequent growth and multiplication. Although the human body contains a plentiful supply of iron, the majority is not easily accessi- ble to microorganisms. The concentration of usable iron is particularly low because lactoferrin, transferrin, ferri- tin, and hemin bind most of the available iron, and the free iron remaining is far below the level required to sup- port microbial growth (81). Thus, microorganisms have evolved a number of mechanisms for the acquisition of iron from their environments (83). Microorganisms pro- duce siderophores that chelate iron with a very high af- finity and that compete effectively with transferrin and lactoferrin to mobilize iron for microbial use. In addition, some microbial species can utilize host iron complexes directly without the production of siderophores. For example, Neisseria species possess specific receptors for transferrin and can remove iron from transferrin at the
- 33. Other documents randomlyhave different content
- 34. Once Carmen's Frenchmaid, suspecting, perhaps, more than a purely altruistic intention in Gwendolen's persistent offerings, warned her young mistress against immoderate indulgence in sweet foods, and protested, with many gesticulations and a hint of tears, that the very last importation of Paris gowns already needed the letting out of seams, and would soon be unwearable. "Nonsense, Lizette," smiled the pampered one, "not eat dulces? I have always eaten dulces. How, in the Virgin's name, would one get through a novel without a plate of dulces beside it?" The maid sent a hostile glance to Gwendolen, which the blonde beauty had the conscience not to resent. Rapidly increasing embonpoint was Carmen's one menace to beauty. She had already begun to pray to her patron saint for diminution. On the prie-dieu invariably lay a half-nibbled chocolate. Were not Gwendolen's friendship so open, so obvious, one might have suspected that she connived with fate to circumvent her Carmen's petition; that actually she assisted in the mournful process of burying perfect features and luscious, languorous dark eyes in warm cushions of pink fat. But no, we must not think such things of Gwendolen. Because of the new intimacy and an increasing activity in Tokio society Gwendolen now saw much less of her schoolmate, Yuki. Perhaps it was as well. The Princess Haganè had her own lessons to learn, and they were Japanese lessons. Following close upon her first sewing-meeting came Yuki's presentation to Their Majesties. The court ladies welcomed her into their midst. As in humbler Japanese circles she was immediately asked innumerable questions. In return she began learning, from her high-born interrogants, the new language of extreme court ceremony. Another reception and another sewing-meeting fell due. To the latter of these functions a mere handful of foreign ladies came. Gwendolen and Mrs. Todd were detained, actually, by some globe- trotting Washington associates, who landed that very day at
- 35. Yokohama. In thetwo subsequent gatherings foreign attendance ceased altogether. Each reception was, however, a "crush." Gossip is a magnet; the presence of eligible young men not exactly detraction. Mrs. Stunt and others of her kind went openly to see whether Pierre Le Beau would attend, and how he would conduct himself before host and hostess. It was the secret craving of such social vultures that a scene, the more disgraceful the better, be enacted for their entertainment, and the disappointment was correspondingly keen when neither Pierre nor Count Ronsard attended. The count, indeed, sent cards and a gift of flowers. No mention at all was made of the younger man. Three of the Haganè official functions had taken place. March hurled itself gruffly into the outstretched arms of spring. Gwendolen knew why Pierre stayed away and why Ronsard remained so impassive. She had good reasons for not telling Yuki. At her friend's silence the latter wondered. Instinct told her that there was a deeper explanation than mere forgetfulness. More than once she had nerved herself to inquire; but always, just on the point of asking, something had happened to interfere. A new cry, which affected Yuki far more openly, began to ring through the current press. "If complications have arisen in Manchuria let Prince Haganè go and unravel them!" This demand grew in insistence with each day. Presently the whole nation had arisen, and was clamoring, "Send our War Lord, Haganè, to the front!" Yuki waited patiently for her husband to inform her of the reception of this demand in high quarters. Like a good Japanese wife she dared not force the issue. On every side her part, it seemed, was to wait, to command herself, to endure suspense. To an impatient nature such as Gwendolen this would have been torture. To Yuki, trained through centuries of brave ancestors to play her woman's part of uncomplaining quiescence, the strain was not so great. Her ignorance of Pierre seemed, indeed, the heaviest burden.
- 36. She scanned nowthe English columns of every paper, hoping against hope that her eyes would seize the printed assurance of his return to France. This was the young wife's prayer, uttered on her knees each night, muttered through pale lips a hundred times each day, that Pierre would go quietly home, and in his own dear land forget the woman who had broken faith with him. His threat against Haganè's life did not sound to her absurd. It re-echoed to her, always with a pang of fear. Love and hate alike give preternatural insight. By injury to Prince Haganè alone could Pierre gain full revenge. By this means he could strip the flesh from the bones of her loyal sacrifice, laying bare the grinning skeleton of a national disaster, wreaked through her. Of course she could not speak these fears to Haganè. There was no one, not even Gwendolen, to whom she could whisper them. Haganè was now seldom at his home. She gathered, once or twice, from gossip of the servants, that he had spent the previous night and day at the Tabata villa, with a small company of statesmen as his guests. In the infrequent visits, she, studying his face with unconscious intensity, saw the same power, the same sadness, the invincible strength unshadowed and unexcited by this renewal of popular hero-worship. The thought that he might leave her alone, to fulfil the duties of his position, brought to the young wife a pang of terror, of misgiving. She believed it to be merely a shrinking from heavy responsibility. To outward appearance she and Haganè stood on opposite shores of an increasing chasm; but in her heart, when she dared listen to its timid pleadings, she knew it to be a narrowing, not a widening, void their joint lives spanned. She could not doubt that he felt some grave pleasure in seeing her on his expected visits to the great shell of his official home. The weekly receptions, where she bore herself with ever-increasing dignity and poise, did indeed give to the husband a deep impersonal satisfaction. It was more than satisfaction that he felt, as he saw the great filled packing-cases sent away each week to suffering soldiers in Manchuria.
- 37. Once, coming inupon her unannounced, as was his custom, he had suddenly taken the white thing in his arms, thrown her head back to his shoulder, and gazed into her eyes as though to drag from some hidden depth an awakening thought,—a cradled possibility. Yuki's lids drooped under the blinding force of his look. She felt as though a great silent wind blew, pinning her against a rock. Surely in his twitching face was more than a calm self-congratulation! It was the man, the master, summoning by right what was rightly his. Love —strong, terrible, yet tender, showed for an instant in his dark eyes. He went from her as quickly as he had come. No word had broken the silence. During the rest of that day Yuki rocked in her heart a new-born hope, a possibility so strange, so ineffable that she dared not open her eyes to its tiny face. With bowed head and fast-closed lids she hushed it. That day set her feet on the temple-stair of shining prophecy. But how dare she, already to one pledge so faithless, climb upward, even on bleeding knees, to that splendid portico above? April spread her witchery of green and flowers over a thousand barren hills. Wild azaleas, wigelia, and bokè (pyrus Japonica) barred the slopes with pink and crimson radiance. Valleys, so lately brown, spread now a wide bloom of violets, a curdled residue of purple morning mists. Earth-dwarfs, congeners of Loki, who people the under-world, drove upward from their subterranean caves huge copper spikes of young bamboo—ten inches across, some of it, as it pierced the mould—a marvellous springing column climbing by joints, two feet a day, toward the sun, and casting off brown sheaths, like outgrown jackets. Children roamed the hedges, the rice-field dykes, and copses (forgotten and unbuilded, sometimes in the very heart of Yedo) for tsukushimbo and the yellow chrysanthemum. All gardens, even those amorphous products of Eurasian uncertainty surrounding the American Legation and Yuki's official home, needed to be fair. Birds came to them, and early butterflies. The sun poured down upon them in equal measure his golden cataracts of joy.
- 38. Saturday of thefirst week came. Pierre Le Beau had not been mentioned to the Princess Haganè, nor had she found a printed notice of him containing a hint of information. Cleverly insulated wires of venom, it is true, attached to her name and Haganè's. Sometimes Pierre was subtly referred to, but never openly. Next day, thought Yuki, she would go to church. Perhaps something would be said of him by the ladies who always crowded so eagerly about her carriage door. This weekly service, in the Episcopal church at Tsukijii, formed now the closest tie that bound Yuki to her Western memories. It was anticipated with eagerness. This link, at least, she told herself should not be snapped. Haganè's consent that she continue openly her Christian devotions had been unqualified. The mail that Saturday morning proved unusually large. An American mail-ship was in. Several letters and papers came from trans-Pacific friends, a great many Tokio social invitations, a few notes relating to Red Cross matters, and one folded pamphlet with a Japanese postmark. She knew from its pink wrapping that it was "The Weekly Hawk's Eye." With a slight shudder she put the evil thing aside, with a vague reawakening of the intention to burn it unopened. Slowly she read her letters and invitations. She glanced through the few American papers for any blue markings. All were finished. She leaned to gather them up and have them taken to her private desk upstairs, when the sun, pointing one bright finger through a blind, fell upon the pink wrapper and rested on her name. "Princess Sanètomo Haganè." It looked very cheerful and suggestive. The dull pink of the cheap paper glowed into a rosy hue. Perhaps it was an omen. Perhaps if she were brave and opened the sheet boldly she would find, instead of the usual malicious innuendoes, the announcement that Pierre was leaving for France. Thinking of Haganè's eyes as they had probed her own she flushed, trembled a little, and murmured aloud, "Oh, if he would only go—if Pierre would only go—how happy—" She broke off. A wave of compunction, pity for Pierre, scorn of her own fickleness, rushed upon her. She took the paper hastily, set her lips for what might be in store, and opened at random.
- 39. Her name wasplain enough, and Prince Haganè's. This time headlines had been dared. "Prince Haganè soon to leave his young wife. The Nation demands his presence at the centre of martial differences. Haganè loath to leave his young wife. Who knows what may happen? M. Le Beau raving in delirium at the German hospital in Yokohama." So much she read and paused. Very quietly she folded the paper and slipped it within a gray silk sleeve. She stooped for the crumpled pink wrapping, smoothed it also, and dropped it in her sleeve. Next she gathered into a neat package the mail she had been reading, rang for a maid-servant, and sent the mail up to her boudoir. Her orders were given in the usual low, pleasant voice. In closing, she said, "Should visitors come I am to be found in this room." Again alone, she walked to a western window and stared out at the great square shadow of the house thrown across the awkward garden. Beyond the straight line of the shadow, paths shone brilliantly in the sun, and flowers danced. Spring had come a little early. Everything that had a blossom to show rushed, it would seem, to the perfumed exhibition. Yuki shivered slightly. For the first time she knew that her hands were growing cold. She moved slowly toward the fireplace, an ordinary foreign grate with coal fire burning. Nearer the warmth she drew out again the pamphlet, unfolded and deliberately read the article from the first word to the last. Some passages she dwelt upon, extracting to its full flavor the bitterness of frustrated hope. According to the "Hawk's Eye" correspondent, Pierre had caught germs of malignant malaria, perhaps of typhus, while wandering in a state of great mental agony along the moats that border a certain official dwelling. He was now at the crisis of his malady. Two nurses watched him night and day, for his dementia had made of him a cunning schemer, full of sly efforts to escape. When detained he raved fearfully, saying that he had "things to do." "The Hawk's Eye"
- 40. ingenuously marvelled asto what these "things" could possibly be. As is usual with articles so inspired the suggestions were far more damaging than any actual statement. She let her hands fall limp. One still clasped the ugly journal. Only a few moments before she had accused herself of heartlessness toward one she had wronged. In her generosity she had almost demanded a deeper suffering, if only it could be directed personally to her offending self, and not include, in its consequences, that great man whose name she now bore. Well, here was her punishment,—a fetid, scalding stream of venom, hurled full and straight at her. Attacks like this were, she knew, less to Haganè than the mud children throw against the base of a lofty statue. His mind moved in a stratum far above such contamination. The nation spoke direct to him. His ear was for his Emperor, the old gods of his race. "Yes," thought the young wife, "I wished to suffer for the wrong I have done, but these writhings of a polluted personality can scarcely be dignified by the name of suffering. It is as if one went forth bravely to combat a knight in armor and encountered a filthy swine. One cannot retaliate upon a beast. Nor,"—here, with a nervous transition to energy, she tore out the offending page,—"nor can I, being his wife, attempt punishment for this defilement." The sound of tearing paper soothed her. One by one she snatched the sheets, crumpling them loosely, and threw each in turn upon the coals, where it twisted, opened its angles, caught in a little puff of smoke, and burned quickly. A sound came to the front door. Some one opened it. She gathered the remaining pages, rolled them hastily into a pithy sphere, and tossed the whole mass to the grate. A soft explosion of smoke and brightness followed. Red light fawned upward to the slender gray figure and excited face. A door of the drawing-room opened, and the draught pulled out from the grate before her a long, pliant tongue of flame. She felt Haganè catch her backward. "That is a risk, to burn papers in these great, ill-constructed chimneys, my little one," he said. Yuki clung to him, staring up into his face to try to judge whether he had already seen the offensive article. He had an unusual animation. She even fancied that his voice
- 41. shook; but itwas not the excitement of anger or disgust. Some national crisis had come. His next words proved the truth of this supposition. "I wish you not cremated this day of all days," he smiled, trying, as she could see, to speak with some lightness. "I need my wife. An opportunity for service has come, more important than all that has gone before. Are you ready, my Princess?" "Lord, I live but to serve you and my land." "We are in a national crisis, Yuki," said her husband. He began to walk up and down the long room with an abandonment to agitation which she had not seen in him before. "A crisis," he repeated. "I shall not explain the matter of it. You need not have the weight and burden of such knowledge, but you can aid me greatly." He paused now near a window. Yuki followed. "I await your pleasure, Lord," she said. He turned to her the deep magnetic gaze she dreaded, yet, strangely enough, longed, at times, to provoke. One massive hand leaned on her shoulder. She had no impulse now to shrink from him. She longed to cower against the strong defence of him, to hide in his breast, in his sleeves, as the frightened souls of little dead children hide in the sleeves of Jizo Sama. As though understanding the unspoken longing he drew her very near. His words were still impersonal. "Some terrible, hidden things long suspected have come to light. I do not believe the wrong past mending. The first step in restitution comes to-day. It is a secret meeting here, in this house,— a small gathering of statesmen, but it may mean to us defeat or victory." "Yes, Lord, I listen. A meeting at this house." "It must appear to be a casual assembling. No servant, not even the good Tora, is to be trusted. When I have given you full instructions I return at once to the palace. Should any unforeseen chance call me back before the hour of one, I charge you speak no words into my ear, nor seek to deflect my thoughts from their
- 42. ominous course. Ibear a heavy burden, Yuki. But the Gods will aid me in my strength." "I will not honorably accost or fret you, Lord." "The statesmen,—and here are the written initials of their names,"—he drew a small scrap of paper from his sleeve—"these seven statesmen, including Sir Charles Grubb and Mr. Todd, will be ushered as usual into these drawing-rooms. If no other guests be present, say to these men in turn, after the first salutations, these exact words: 'I have received from my lord instructions and the initials of your name.' Can you repeat precisely?" Yuki did so. "That is well. Thirteen words, remember. They make to these seven a sort of password. Each, as you speak, is to be conducted to my small office-room to which the wooden doors, and the heavy portières also, are to be drawn." "I understand, your Highness. But what am I to do if other visitors come?" "Ah, little Princess Haganè, it is in such straits that your experience of foreign social hypocrisy must be made to serve you. It is of imperative need that you do not leave this room after the hour of the Rat (1 p. m.). Yet it is also imperative that you receive, equally, all guests. Those unbidden you must get from the house." "It is a difficult task, Lord, but it may be done." "That is a brave wife. Remember that not only from the time of the Rat, but this hour, too, this very moment, commences your vigilance. Tale-bearers and enemies may be lurking near. If human ingenuity can keep a meeting secret this will be kept, but, alas, in a time of great issues the dragon's teeth sow spies instead of men. Do you understand all I have said, my Yuki?"
- 43. "I understand, yourHighness, and am honored to do your august bidding." Before leaving her he gazed for another moment steadily into her upraised face. "You are pale to-day as your name, my small snow-wife; yet your eyes move and glitter with a strange unrest." "I beseech your Highness concern not your weighty thoughts with my unimportant outer appearance." "I must not do so, indeed," murmured her husband. "My chief thought now must be my Imperial Master. Farewell, little one. I shall arrive at one, if not before." Yuki followed him to the door for a last wifely obeisance. The carriage had been waiting for some moments. After the loud rattling of wheels came a hollow silence. Yuki stood on the granite doorsteps looking outward with unseeing eyes. The house-shadow shrank closer to the huge cube that cast it. Sunshine, like a golden fluid, brimmed up the azure walls of day. From garden-beds nearby, and from path-borders leading into hazy distance, blossoms beckoned. She saw only an iridescent blur. The jinchokè (called by foreigners Daphen Odora) rose in waxen masses of white or arbutus pink. Azaleas heaped formless hillocks with Tyrian hues, and the long yellow sprays of yama-buki, to which Gwendolen had so often been compared, poised waiting for the breeze, or else tossed in bright indignation at the sudden desertion of a bird. Sweet odors flowed inward, and whispered her to follow. Still half unconsciously she stepped down to the gravelled path and began to walk in the garden. Sometimes, among the beautiful familiar blooms, an alien flower smiled, a budding rose-tree, or a purple blotch of English violets. The thought of Pierre's danger came now with less of acid pain. Perhaps this illness was to save them both—and Haganè. The long hospital days might bring to the young Frenchman clearer judgment, and perhaps a more forgiving heart. In convalescence, surely, he would wish to return to his own land. At such times the spirit is fain
- 44. to leave theweak body, and speed on before, to childhood's home. She had reached a cluster of the early iris. These were Pierre's flowers, the lilies of his France. She stroked the silken petals as though they were hands. "Pierre, my poor, poor Pierre," she breathed aloud. "My Yuki-ko," came as an echo. Yuki started and looked around in fear. "Little flowers, was it you that spoke my name?" "Yuki," came the low voice again. "Do you grieve for Pierre? Poor Pierre is dead!" He stepped out from behind a cluster of dark cypress-trees. Yuki bit her lips to keep from screaming. Was this the ghost of the man she had loved? "Yuki," said the phantom, with a little chill whine in his voice, "won't you even speak to me?" "Is it you, Pierre, or is it indeed your newly fled spirit come to reproach me?" Pierre ran his hands through his short, dry hair, then dropped them, as if the effort had been too great. He took a step forward. "Why, yes, it is Pierre, after all. I thought I was dead, but I am not. Yes, sweetheart, you may come to me. It is your Pierre." Yuki ran to him and caught one dangling hand. It burned her like hot metal. "You escaped, in spite of your two nurses?" she cried. Pierre began to whimper. "Yes, yes, Yuki, I got away at last. I had things to do. Don't send me back there, Yuki! My room has bars, like a cage." "How did you get away?" "Little Jap nurse couldn't resist me. Told me of a back entry. Nice little nurse in white cap. Jap—cap; cap—Jap. Ha—ha!"
- 45. "Come, dear," saidYuki, pulling him gently. "I will not send you back. You shall go with me to the little Cha no yu rooms at the far end of this garden. There you can lie down until you feel better. Will you follow me quickly and in silence along this little path?" She pointed. "Indeed I will—no need to ask twice," cried the sick man, and began to giggle like an excited child. "I'd follow you anywhere, Yuki. Are we running away to be married?" "Hush, Pierre; if you laugh and speak so loud others will hear you and send you back to prison. We must be very, very quiet." "Very quiet," echoed Pierre, solemnly. "Never do for old prince to hear us, oh, no!" He began to mince along on the tips of his toes, giggling every now and then at the thought of the trick they were playing. Yuki sped on before him, like a fawn. At the tea-rooms she sprang to the narrow, railless veranda, drawing a single shoji panel carefully to one side. The two small rooms were in order. Sunken into the floor of one was the copper hibachi, two feet square and now filled with cold ashes, an article indispensable to tea-rooms of ceremony. The sun pouring against translucent paper walls flooded the small space with radiance. "What dear little rooms!" exclaimed Pierre, as he scrambled in, panting. "She would call them 'cunning little rooms,' that yellow- haired American girl. What was her name, Yuki? She is not a good friend to poor Pierre; she could not swear it when I asked her. Are these the little rooms where we are to live, Yuki, now that we have run away from the old prince and are married?" "Yes, dear," said Yuki, soothingly. "Here is where Yuki will care for you until a betterness comes. See, I shall heap for you these nice cushions. They are your Japanese pillows. You must lie on them very
- 46. still, and keepall these shoji shut close until I can go and get some medicine for you." "No!" said Pierre, fractiously. "Medicine no go! Kusuri, ikanai! Too much kusuri every day at hospital. Nurses all carry spoons in their belts. I don't need more medicine, Yuki; only for you to kiss me. You haven't kissed me all day!" He threw himself among the bright cushions and began tossing his head from side to side. "I will kiss you when I get back," said Yuki. "Only promise to lie here very quietly until I can come, and many times I will kiss you." Pierre raised himself on an elbow and looked dubious. "Kiss me before you start," he demanded. "You break promises, you know. And this morning you have such a droll fashion of going suddenly far away, and then starting back quickly, just like the end of a trombone that one is playing. You must be a witch, Yuki, to move so swiftly through the air. Kiss me, or I shall not believe it is really you." With a heart strained to the limit of endurance Yuki knelt beside him on the matted floor and pressed her ashen lips to the red coal of his mouth. Pierre, seizing her with superhuman strength, kissed her again and again, until the tortured woman felt that she must rend the air in clamor to some native god or demon who might save her. This passion, branded on the soul of Prince Haganè's wife, gained a new and terrible power of defilement. In a spasm of anguish she wrenched herself free, went backward from him, and seized the shoji's edge to hold herself. "I will kiss you no more until you take the medicine," she said, with a steadiness that surprised them both. He lurched forward, grasping at a swaying sleeve. She eluded him. "If you are not more controlled I will leave you altogether, and send police to take you back to Yokohama!" He grovelled at her feet and whimpered. "I'll be good. Don't send me, Yuki. But if I lie quite still you'll kiss me many, many times again when you return, won't you?"
- 47. Yuki hesitated. Hedragged himself half upright. "You shall. I'll kill you! I'll kill myself, here! You must kiss me. A wife always kisses her husband. Swear that you will kiss me!" The light of increased madness glared in his beautiful eyes. "Yes, I'll kiss you, I swear it," faltered the girl. Pierre laughed foolishly in his satisfaction. "Then I'll lie still among your pillows, little wife. Old prince sha'n't find us. Put us in boiling oil, that old prince. Don't be gone too long, little wife." Yuki hurried along the intricate paths toward the house. Dry sobs rose one after another slowly, coming relentlessly upward in her slender throat with a distention that grew to agony. "I must not stop to think, I cannot give up now," she panted. "O Kwannon Sama, what am I to do?" This black hour, like some dark chemical, was turning the memory of all other grief to light. The one conscious thought which her mind hugged jealously was Pierre's necessity for medicine. Fortunately, she knew a little of this, and kept a well-filled chest. His fever was terrific. Human pity demanded that she first allay this raving torment of the blood before delivering him to cold officials, or even to Count Ronsard of the French Legation. Her thoughts and plans in this present bewilderment could get no further than the fever-draught now to be given the sick man. With shaking hands she prepared it, and then a second drink, a powerful sleeping- potion. She got back to him as noiselessly as she had come. Apparently no one had seen her. Pierre was now in actual fever- madness. He had thrown coat, waistcoat, and watch in various parts of the room. The cushions were strewn wide. A corner of one rested in hibachi ashes. In one of his hands he clasped tightly the half of a long ivory hairpin. With the patience of a mother and the ingenuity of a wife she coaxed him, at length, into swallowing one of the draughts. He did not demand the promised kisses. He did not know her now, or, rather, the recognitions came in short flashes, like heat lightning. Sometimes he took her to be Gwendolen and accused her angrily of
- 48. connivance with Haganèand the ambitious Onda family. Again he thought her the German head physician and raved of his wrongs. He passed rapidly from one language to the other, essaying at times his broken Japanese. It was generally in English that he denounced his faithless sweetheart, and the epithets directed against her caused Yuki's heart to sink with shame,—not for herself, but for him. A longer interval of sanity came. He recognized his companion with piteous little cries and tears of joy. He believed that at last they were married, and prattled on of the long, happy future, of their little home in France, until Yuki, having come for the moment to the end of suffering's capacity, listened with a dreary smile and dull ears. The second draught, the sleeping-potion, was to be given in half an hour. Through that interminable time she waited, his head upon her aching knees, his fevered hands reaching ever for her face, her shoulder, until lethargy alone saved her from an answering insanity. The plan was half formed in her dull thoughts to administer this potion, then, when slumber overcame him, to close the shoji, and leave Pierre to sleep away the fiercest fever while she could think out a way of getting him from the garden. But for the political meeting, falling so strangely on this very day, the situation would have possessed no great peril. It would have been merely a sick man who, in delirium, had wandered unknowingly into Haganè's garden. The servants might have found him; Ronsard have been telephoned for, and Prince Haganè himself asked what was best to do. This was what might have been; but here was the matter as it really lay. A Frenchman, and attaché of the Legation,—ill or well no less a Frenchman—concealed in Haganè's garden, sheltered and protected by Haganè's young wife! Yuki gave a convulsive shudder. The sick man gasped, and clutched the air as if he thought himself falling from a height. Fate smiled a thin, hard smile down into Yuki's eyes. The girl did not resent Fate's prophetic stare. Already she knew herself trapped. Her wild thoughts had run since the beginning of
- 49. eternity in thissame ring of fire. There was time for nothing. The one frail chance was that Pierre should sleep on through the meeting undiscovered. Already twelve o'clock had come. From the high land near the samurai Onda's home, a big bell boomed and quivered out over the city. The echoes stirred and shifted tranquil layers of the noon. Fear sank down like soot upon a crouching woman with the sick man on her knees. Pierre, for some moments past, had gradually ceased the restless tossing of his head, and was forgetting to utter short, disjointed words. The fair hair, that had been so stiff and dry, clung now in moist locks about his temple. His delicate hands ceased twitching and picking at Yuki's gown, and fell over limply on the floor. Caught loosely in the right hand lay the broken hairpin. To any Japanese, of any class, this would be fatal evidence. Under her fairy-like touch he gave a start, clutched more firmly at the pin she was trying to take, and threw his hand upward above his heart. Again Fate smiled, and Yuki bowed her head. Now a soft, regular breathing began. The healing sleep was on the sufferer. His face was growing young and gentle. Yuki stared down into it, tearless. Her heart, like some living entity beaten and tortured too long, had lost the power of sensitive response. There was only a dull, incessant aching that was becoming, already, an acknowledged part of her. He was safe. To-day's crisis, at least of the devouring heat, was over. He would awake refreshed and clear. As for her, everything had grown so vague and far-away she cared very little what might happen. The insensibility of reaction bore her outward on a warm tide. Danger lost its meaning, and grew but a shadow-play on life. A Frenchman in Haganè's garden, and a crucial meeting to go on in the house! There was something piquant, fetching, in the idea. Yuki nodded above it and smiled. Oh, she was so tired, so tired of everything! A little malicious something was tapping, tapping, just at the base of her brain. The ache at her heart benumbed her. A desire, dull and insistent as the pain itself, crept to her, just to lie upon the matting near poor Pierre and rest. They belonged together, the weak
- 50. ones. Chance anddisappointment had thrown them about like toys. What had such as they to do with the God Haganè? Yes, she had better fail once more, and it would be the last. Let the grave statesmen come and go, let Haganè seek her! She had nothing to do but the easiest of all things, just to do nothing, and all this benumbing misery would be at an end. She wondered, still smiling, in what way Haganè would kill her. She fingered curiously the stops of a dozen fearful thoughts, and felt no fear. Had law permitted him to carry the two swords of his class, the short one would deal a quick and merciful death. Since he was unarmed perhaps he would simply let one of the servants slay her, not caring to soil his hands with such feeble stains. An influence was coming over her in rhythms, like tepid waves. A delicious lightness blew upon her brain. She gasped for insensibility as for music, dumb, perfumed music, drunk in by pores of the flesh. One small nerve of desire began to tingle. "Oh, let it go on," she cried to her soul; "have no interference! Let me pass into nothingness by this heavenly gliding!" As from a great distance came footsteps and the sound of commonplace voices. Yuki moaned aloud, and crept an inch nearer her companion. "She was seen last coming in this direction," said a speaker; "Ii, the gardener, saw her." "She is not in the adzuma-ya! Can it be that our gracious lady has gone for repose to the tea-rooms?" "Baka!" exclaimed the other whom she now recognized as Tora, the butler; "is not that great official residence sad enough and lonely, that the poor child seeks a more desolate place? I pity her." "Luncheon becomes honorably cold upon the table," murmured the boy, showing compassion in his own way. "And foreign food
- 51. when chilled, withthe grease becoming as wax about the edges, is of all sights the most disgusting." "Arà," sighed Tora, "she eats little enough even when the food is hot." "Those many disgraceful things said of our lady in the newspapers,"—the younger servant was beginning, when Tora stopped him fiercely. "Gossip not of your betters, boy! You should not read such things. There are no truths in printed scandals. Come, not that way, she is not in the tea-rooms. I see a fresh disturbance of the gravel along this path." To the listener's intense relief they turned sharply to the left. Wide awake now with an intensity of sensitiveness that made every stirring leaf an enemy, the young wife crept outward from between two shoji, closing them with the extreme of care. In full sight, on the veranda, lay her little foreign handkerchief. No other woman on the place used lace-bordered handkerchiefs. Tora must have seen and recognized it, and, in an instant, perhaps, of protection, have led the boy aside. Yuki's cheek burned. She dared not think Tora's thoughts. This humiliation was a wound made with a weapon of poor metal, yet she could not, even then, refuse gratitude for the delicate consideration. As the two servants came again into the main part of the garden, their mistress walked quite leisurely a few yards before, stooping now and then to a flower, or gazing up with smiles to a blossoming cherry-branch. "Luncheon is served, your Ladyship," said Tora, gravely, and bowed before her in the path. "I will come immediately," returned Yuki. She did not meet his eyes.
- 53. CHAPTER TWENTY-FIVE During theshort, uncomfortable meal Tora stood like a painted stake behind his mistress's chair. The "boy," attempting to supply the watchful efficiency his senior for once appeared to lack, kept his small eyes darting from her white face to the "dirty wax" at the edge of her plate, until Yuki thought she must deliver herself over to an attack of laughing hysterics. Tora poured and brought her wine unbidden. Again she resented his presumption, again felt a cowed sense of thanks for his solicitude. Abandoning the table at the first possible moment, she went swiftly upstairs to her own chamber and rang for the maid. The simple morning robe of smooth silk must be changed for a more elaborate afternoon toilette. She selected a curdled gray crêpe with tiny silver pine-leaves sprinkled through it. The under-robe was turquoise blue; her wide sash of blue-black satin brocaded in conventionalized silver pine-branches. The transfer went on with breathless celerity, yet the hands of the mantel clock moved faster still. Ten minutes only lacked to the hour of the Rat. The sound of carriage-wheels crunching gravel rose from the drive below her. Yuki gave a restless motion of her entire body, and turned her face around to the maid, who now tied the great loop of the sash. "Patience an instant longer, your Ladyship," smiled the maid. "Let me but girdle your illustrious person with the obi-domè and I shall be done." "Here is the obi-domè," cried Yuki, her voice betraying her impatience. "I shall retain one clasp while you wind it around the sash." She took up from among the American toilet articles on her
- 54. dresser the articledesired, a flat, soft braid of silk with golden clasps. Yuki, as she had said, held one end against the front of her sash, while the maid dexterously threaded the high sash loop at the back, and brought the answering clasp to its mate. It clicked like an old-fashioned bracelet. A servant knocked on the door. Yuki herself answered. With mingled relief and perturbation she read on the cards the names of Mrs. Todd and Miss Todd. It was an unfortunate time for their visit, yet now as always the thought of Gwendolen's presence brought a little stir of excitement, a sweet glow of true happiness. During her flight downstairs Yuki formed the clearest resolution that had come to her in the distracting day. She would tell Gwendolen of Pierre's presence. If help were possible, Gwendolen would find a way. The new hope brought a little glow to the face which greeted her American friends. A little talk on unimportant, pleasant matters would refresh and steady her. For a moment only did the bright illusion abide. Gwendolen and her mother bore, in common, an air of hesitating excitement. "Oh, what is wrong now?" cried Yuki to them both. "Well, you are quick!" said Gwendolen; "have we become mere transparencies, or do your wits acquire a preternatural alertness in these big rooms? Yes, there is something wrong—not fatally so, only a menace." "We felt it our duty, Yuki—" began Mrs. Todd, on her lowest register. "Now, mother," Gwendolen interrupted, "you promised faithfully to let me tell Yuki in my own way. You sound as if you hooted from a cave. It isn't anything horrid, darling!" This last speech was directly to the princess. "Don't begin to fade away. It is simply that Pierre, who has been ill at the German hospital in Yokohama, escaped this morning, in delirium, and the authorities are after him."
- 55. "In delirium—raving indelirium—the poor tortured boy!" echoed Mrs. Todd's sepulchral tones. "Oh, is that all?" breathed Yuki. Her face showed unmistakable relief. Gwendolen stared at her, incredulous. Mrs. Todd put up her lorgnette. "All! Did I understand you to say all? Is it not enough? Have you known before to-day of his terrible illness?" "No, indeed, I have not, dear Mrs. Todd. And by 'all' I did not mean the heartlessness, as you think. I only meant—I meant—" "Humph!" said the matron, suspicion deepening with the sight of the young wife's confusion. "Perhaps Pierre has been here already. Has he been here, Yuki?" Yuki looked more embarrassed than ever. She hesitated the fraction of an instant. Gwendolen's eyes sent out one hazel gleam. "No, dear Mrs. Todd," answered Yuki; "Monsieur has not set foot in this house since my first reception, many weeks ago." "Humph!" said Mrs. Todd again, and closed her lorgnette with a disappointed snap. "Well, there's time for him yet! You had better look out, for if he is found here—" She shut her lips with a snap like the lorgnette-case. Because of avowed sympathy with Pierre, the good lady had assumed an air of displeasure with Yuki which all the new rank and wealth could not overcome. Yuki, strange to say, liked her the better for it. She hugged the memory of Mrs. Todd's cool looks as a fanatic might have hugged his haircloth shirt. Gwendolen had turned away. She did not wish either Yuki or her mother to gain a hint of her personal thoughts. At Yuki's last statement, her quick mind had supplemented, "He has not set foot in this house. No—but the garden is wide, the steps and galleries inviting." Yuki hid some gnawing secret, of this she was sure. More carriage-wheels crunched the gravel and Yuki's heart at once.
- 56. "Ah," said Gwendolen,coolly, now beside a window, "here's the Emperor come to see you, Yuki!" Yuki ran forward gasping. Anything might have happened on this reeling day. "No," laughed the other. "I just teased you. But it is some magnate, I assure you. My heavens, what a swagger!" Mrs. Todd, hastening to her daughter's side, drew the window- curtain farther. Her face glowed with satisfaction. "Prince Korin," she announced, "he is a dear man! I shall be pleased to meet him again." "Come along, mother," said Gwendolen, a little brusquely; "he hasn't called on us." "I sha'n't do anything of the kind," said the matron, indignantly. "Prince Korin took me in to dinner last week at the German Legation. Doubtless he will be as much pleased as I to renew the acquaintance." "Please do not urge your mother to depart," Yuki flung back over her shoulder as she went toward the door; "I want to speak with you, Gwendolen, on some important matter." Without a qualm she delivered the wondering peer into the outstretched hands of the American lady. Drawing Gwendolen to a corner of the big room she said, in a low and agitated voice, "He—that one we spoke—he is even now asleep in this garden. It is terrible, but I could not send him off. I gave medicine; he was nearly to die of great illness. Make no sound or look of surprise; no one suspects, unless it is the butler, Tora. Perhaps you can help me. What makes all more dangerous, more terrible, is a secret meeting of state to be held here this very hour. Prince Korin is the first. You and Mrs. Todd must go before Haganè come, or he will feel great anger to me. Your father is to arrive. Oh, Gwendolen, do you see any way to save?"
- 57. "It is themost frightful complication I ever knew in my life," said Gwendolen, awed for once into calm. "Why, of all days, should the meeting fall on this?" "Some terrible crisis in war. All may depend on this hour,—our very national existence." "I knew something was up. Dad is cross as a bear, and Dodge struts like a turkey. Yuki, there is but one thing. Your husband must be told the moment he enters this house!" "Oh, if I could do that!" cried Yuki. "No such tearing thoughts could I have felt. But he has given orders to me not to disturb his mind on anything until this meeting has passed." "Nonsense, you must disobey of course," said the other; "unless I myself could get Pierre out of the garden." Her practical American wits worked rapidly. "I can do it I think. You must have smaller gates to these high walls." "Yes, yes, on all other days," said Yuki. "But not just for this one day. Everything—everything—for these few hours are bolted. I think it to be karma, Gwendolen. No use to fight for me!" "Now look here, don't go into despair so soon. You say you gave medicine. Is it a sleeping draught?" "Yes, first the strong fever-cure; then, half-hour later, a sleeping potion. It is strong. It would keep the Japanese asleep for many hours." "Go to your husband, Yuki. You must do it; never mind disobedience!" "But if some strange thing that you, not being Japanese, cannot foresee should hold me back, do you think there is other chance?"
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