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- Herausgeber: Wiley-VCH
- Kategorie: Fachliteratur
- Sprache: Englisch
All-in-one guide to monitoring and maintaining microbiological safety in the manufacturing of pharmaceuticals, diagnostics, and cosmetics
Addressing the full spectrum of microbiological quality control and quality assurance in pharmaceutical production, Pharmaceutical Microbiology covers methods and technologies required by regulatory authorities throughout the world, with all methods and protocols rated in terms of their compliance with current (2023) EU legislation.
Written by the former head of biological quality assurance for one of Europe’s biggest pharmaceutical and diagnostics companies, Pharmaceutical Microbiology covers sample topics including:
- General conditions for the operation of microbiological laboratories, calibration and qualification of devices, and type culture maintenance
- Industrial hygiene, ambient monitoring, quality control, process validation, microbiological water examination, and rapid microbiological methods
- Automation in the microbiology laboratory, quality assurance, identification of microorganisms, cleaning, sterilization, decontamination, and disposal, and contract testing
- Pharmacopoeial and non-pharmacopoeial methods for the identification and quantification of microorganisms, including cell culture and selected animal tests
Pharmaceutical Microbiology is an essential practice-oriented all-in-one reference for engineers, researchers, and professionals involved in setting up and running a microbiological quality control unit in the pharmaceuticals, diagnostics, and cosmetics industries.
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Veröffentlichungsjahr: 2025
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
Preface to the English Edition
Preface to the First German Edition
List of Abbreviations
Glossary
1 Introduction to Microbiology
1.1 Historical
1.2 Importance
1.3 World of Microorganisms
1.4 The Bacterial Cell
1.5 Taxonomy of Microorganisms
1.6 Medical Microbiology
References
Further Reading
2 General Conditions for the Operation of Microbiological Laboratories
2.1 Laws and Technical Regulations
2.2 Medical Care for Employees
2.3 Operating Description for Microbiological Laboratories
2.4 Establishment of Microbiological Laboratories
2.5 Nutrient Media
2.6 Media Preparation
References
3 Calibration and Qualification of the Devices
3.1 Calibration Error
3.2 Calibration
3.3 Balance
3.4 pH Meter
3.5 Piston Pipettes
3.6 Stopwatch
3.7 Devices for Achieving Specific Temperatures
3.8 Clean Bench
3.9 Air Sampler
3.10 Particle Counter
3.11 Measuring Device for Determining the Water Activity
3.12 Photometer/Reader
3.13 Tube Reader for Endotoxin Determinations
3.14 Fluorescence Reader for Endotoxin Determinations
References
4 Stock Collection
4.1 Reference
4.2 Shipping
4.3 Storage
4.4 Cultivation
References
5 Industrial Hygiene
5.1 Hygiene
5.2 Microbiological Basics for Hygiene
5.3 Hygiene Measures
5.4 Sterilization, Disinfection, and Aseptic Production
5.5 Hygiene Protocol for Microbiological Laboratories
5.6 Pest Control
5.7 Hygiene Officer
5.8 Implementation of Hygiene Training Courses
References
6 Environmental Monitoring
6.1 Methods
6.2 Microbiological Monitoring in the Sterility Test Isolator
6.3 Physical Monitoring in Sterile Production
6.4 Physical Operation
6.5 Evaluation of the Microorganisms
6.6 Register of Microorganisms
References
Further Reading
7 Quality Control
7.1 Pharmacopeial Methods (Compendial Methods)
7.2 Non‐Compendial Methods
7.3 Tests Using Animal Models
7.4 Cell Culture Methods
7.5 Validation of Pharmacopeial Methods
References
Further Reading
8 Process Validations
8.1 Media Fill
8.2 Depyrogenation
8.3 Validation of Sterilization with Dry Heat
8.4 Validation of Sterilization Using Moist Heat (Autoclave)
8.5 Validation of Sterile Filtration
8.6 Container Closure Integrity Test
8.7 Cleaning Validation
References
Further Reading
9 Microbiological Examination of Water
9.1 Sampling
9.2 Specimen Transport
9.3 Use of the Different Water Qualities (Table 9.6)
9.4 Purified Water
9.5 Highly Purified Water
9.6 Water for Injection
9.7 Water for Diluting Concentrated Hemodialysis Solutions
9.8 Water for the Preparation of Extracts
9.9 Drinking Water
9.10 Legionella
References
10 Rapid Microbiological Methods
10.1 Determination via ATP Content
10.2 Determination Via the Incorporation of Fluorescent Markers
10.3 Flow Cytometry
References
Further Reading
11 Automation in the Microbiology Laboratory
11.1 Automatic Dyeing Machines
11.2 Devices for Counting Colonies
11.3 Automatic Nutrient Media Filling Machine
11.4 Automation of the Endotoxin Test
References
12 Quality Assurance
12.1 Structure of an SOP System
12.2 Training
12.3 Audits and Inspections
12.4 Procedure for OOS and OOE Results
References
13 Microorganism Identification
13.1 Growth Curve
13.2 Generation Time
13.3 Preparation of Pure Cultures
13.4 Sensory and Macroscopic Characteristics
13.5 Microscopic Examination
13.6 Staining
13.7 Principle of the “Colored Row”
13.8 Immunological Procedures
13.9 Polymerase Chain Reaction
13.10 Gas Chromatography (FAME)
13.11 FT‐IR Spectroscopy
13.12 MALDI‐TOF
References
14 Cleaning, Sterilization, Decontamination, and Disposal
14.1 Cleaning
14.2 Sterilization
14.3 Laboratory Cleaning and Disinfection
14.4 Disposal of Infectious Waste
14.5 Disinfection Measures in Case of Accidents
References
15 Contract Testing (
Outsourcing
)
References
Further Reading
16 Microbiological Networks
16.1 CPM
16.2 VAAM Expert Group Quality Management
16.3 Subcommittee Microbiology in the VfA
16.4 DGHM
References
17 Addresses
Literature
Legal Requirements
Basics of Microbiology and Hygiene
Fungi/Yeasts
Cleaning and Disinfection
Detection of Microorganisms
Hygiene Protocol
Microbiological Examination of Water
Environmental Monitoring
Detection of Endotoxins and Pyrogens
Training of Employees
Calibration of Measuring Instruments
Laminar Flow Safety Cabinets
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 Groups of microorganisms and biological agents.
Table 1.2 Sizes of particles and cells.
Table 1.3 Living range for molds. In the area marked with
X
, the growth of ...
Table 1.4 Environmental conditions for microorganisms.
Table 1.5 Minimum water activity values (
a
w
) for the growth of various micr...
Table 1.6 Human‐pathogenic
Escherichia coli
strains.
Table 1.7 Infectious diseases and their modes of transmission, pathogens (c...
Table 1.8 Some human diseases and the probable animal sources of infection....
Chapter 2
Table 2.1 MOPS buffer (3‐(N‐morpholino)propanesulfonic acid buffer) for the...
Table 2.2 Different sizes of Petri dishes and their uses.
Chapter 3
Table 3.1 Nominal volume and error limits for piston‐stroke pipettes accord...
Table 3.2 Defined temperature ranges according to Ph. Eur. and USP.
Table 3.3 Temperature ranges frequently required in the microbiology labora...
Table 3.4 Correction table for the impaction collector MAS 100 NT
®
, which c...
Table 3.5 Compilation of air samplers with information on the manufacturer/...
Table 3.6 Calibration solutions according to USP XL (2017), chapter 〈1112〉....
Table 3.7 Recommended calibration frequencies.
Chapter 4
Table 4.1 Compilation of microorganisms published in the European Pharmacop...
Table 4.2 Storage times of different microorganisms depending on the storag...
Chapter 5
Table 5.1 Microbial colonization of healthy and diseased humans.
Table 5.2 Cleanroom classes according to Annex 1 of the EU Guide to Good Ma...
Table 5.3 Cleanroom classes according to Annex 1 of the EU Guide to Good Ma...
Table 5.4 Monitoring and limit values in critical areas A–F.
Table 5.5 Monitoring frequencies in critical areas A to F.
Table 5.6 Release of particles (skin flakes) per minute by various human ac...
Table 5.7 Comparison of cleaning, disinfection, and sterilization effects....
Table 5.8 Areas of application and methods for disinfection.
Table 5.9 Examples of cleaning agents and disinfectants for various applica...
Table 5.10 Active substances for disinfection of non‐enveloped and envelope...
Table 5.11 Disinfection plan for the microbiology laboratories (example).
Table 5.12 Toxicity of the various insecticides to humans and animals.
Table 5.13 Typical hygienic pests and disease vectors in Central Europe.
Chapter 6
Table 6.1 Comparison of clean room (partial barrier) and isolator (absolute...
Table 6.2 Gaseous sterilization or decontamination.
Table 6.3 Results of glove tests (pressure test and visual).
Table 6.4 Working day monitoring of air and surfaces in the isolator.
Table 6.5 Standard and alarm values of the physical parameters.
Table 6.6 Assessment of criticality of alarms and responses to them.
Table 6.7 Cleanroom classification according to ISO EN DIN 14644‐1:2015. Th...
Table 6.8 Number of measuring points from DIN EN ISO 14644‐1, 2015.
Table 6.9 Listing of air exchange rates (technical recommendation), overpres...
Table 6.10 Microbiological environmental monitoring in physical operation....
Table 6.11 Evaluation of the findings in the environmental monitoring over ...
Table 6.12 Evaluation of personnel monitoring from one quarter.
Table 6.13 New nomenclature for pseudomonads and closely related microorgan...
Chapter 7
Table 7.1 Advantages and disadvantages of the endotoxin determination metho...
Table 7.2 Different endotoxin formulations.
Table 7.3 Dilution table.
Table 7.4 Dilution table.
Table 7.5 Example 1. Geometric mean value = 0.24 IU/ml. From the double det...
Table 7.6 Example 2: Geometric mean value = 0.17 IU/ml. From the duplicate ...
Table 7.7 Substances tested in the LAL test. The MVD is given for the curren...
Table 7.8 Results of three in vitro pyrogen tests with fresh human blood (e...
Table 7.9 Pyrogen test of vitamin D
3
. Acceptance criterion according to Ph....
Table 7.10 Ultrafiltration of the vitamin solution. Duration: 30 minutes; m...
Table 7.11 Test with different endotoxin spike concentrations in the vitami...
Table 7.12 Test with different endotoxin spike concentrations in the vitami...
Table 7.13 Reagents for unmasking.
Table 7.14 Test organisms of Ph. Eur., USP, and DIN EN ISO.
Table 7.15 Preservation requirements for parenterals and ophthalmics.
Table 7.16 Preservation requirements for topicals.
Table 7.17 Requirements for Oralia. The criteria represent the recommended e...
Table 7.18 Summary table for sterile products, ophthalmics, and topicals.
Table 7.19 Example 1 for a preservative exposure test with the test results....
Table 7.20 Example 2 for a preservative exposure test with the test results...
Table 7.21 Properties of selected mycoplasmas [24].
Table 7.22 The most important human and animal pathogenic mycobacterial spe...
Table 7.23 Compilation with the bioindicators specified by Ph. Eur., chapte...
Table 7.24 Test organisms used for vitamin determinations.
Table 7.25 Summary of the results of the tests on cellulose prefilters.
Table 7.26 Microbiological action limits for primary packaging materials fo...
Table 7.27 Measured cell sizes in WfI and CSB after increasing incubation t...
Table 7.28 Bacteriological specifications for the detergents and disinfecta...
Table 7.29 Percentage distribution of laboratory animals in Germany in 2009...
Table 7.30 Compilation of data on the most important test animals.
Table 7.31 Antibiotics for use in cell culture.
Table 7.32 Test microorganisms (only ATCC numbers are given) and incubation...
Table 7.33 Visual growth test of the inoculated test organisms in the filtr...
Table 7.34 Neutralizing measures and agents.
Table 7.35 Test microorganisms and incubation conditions for validation of T...
Table 7.36 Detection of specified microorganisms.
Table 7.37 Sample preparation for the detection of the specified microorgani...
Table 7.38 Evaluation of the quantitative detection of bile‐tolerant, Gram‐...
Table 7.39 Exemplary results after testing for inhibition and enhancement,
Table 7.40 Threshold and maximum dose for different routes of administratio...
Chapter 8
Table 8.1 Formal process validation procedure.
Table 8.2 Bacterial concentrations according to MacFarland standard (accord...
Table 8.3 Depyrogenation in the hot air sterilizer (250°C, 120 minutes) at...
Table 8.4 Results of the runs of ten endotoxin‐doped 10 ml glass ampoules a...
Table 8.5 Loading schemes for two sterilizers. The pipette box contains gla...
Table 8.6 Kill times at temperatures of 100, 105, and 121 °C.
Table 8.7 Results of visual inspection of 300 analyzed primary containers a...
Table 8.8 Determination of the number of proliferating
Pseudomonas aerugino
...
Table 8.9 Growth test of inoculated medium after microbiological integrity ...
Table 8.10 Results (mean values of duplicate determinations) of endotoxin s...
Chapter 9
Table 9.1 Microorganisms frequency in % in water.
Table 9.2 Pathogens that can be transmitted to humans via water.
Table 9.3 Disease outbreaks caused by contaminated water (selection).
Table 9.4 Disinfectants for eliminating microorganisms in biofilms.
Table 9.5 Microorganisms present in biofilms, modified according to Ref. [7...
Table 9.6 Use of the different water qualities.
Table 9.7 Nutrient content of agar media CSA and R2A (both according to Ph....
Table 9.8 Action, warning, and tolerance limits for the different water qua...
Table 9.9 TVO [15] specifies the permitted microbiological parameters in Se...
Table 9.10 Total hardness ranges (in millimole per liter) of drinking water...
Table 9.11 Typical contaminants in drinking water.
Chapter 10
Table 10.1 Summary table of rapid microbiological methods.
Table 10.2
Pseudomonas aeruginosa
cultured in CSB. Determination of turbidi...
Chapter 12
Table 12.1 Health authorities of various countries worldwide.
Table 12.2 State health authorities in Germany, with the highest state heal...
Table 12.3 Evaluation of the test for pyrogens according to Ph. Eur., table...
Chapter 13
Table 13.1 Comparison of light and electron microscope.
Table 13.2 Gram‐negative and Gram‐positive bacteria and their cell shape.
Table 13.3 Presence of different types of fatty acids in different bacteria...
Table 13.4 Wavenumbers of molecules detectable by FT‐IR spectroscopy.
Chapter 14
Table 14.1 Inactivation temperatures and times, related to moist heat.
List of Illustrations
Chapter 1
Figure 1.1 Bacteriophage T4, acceleration voltage 80 kV, uranyl acetate as c...
Figure 1.2 White mold on damp furniture wood in the basement, after rainwate...
Figure 1.3 SEM image of
Aspergillus niger
on agar plate.
Figure 1.4
Escherichia coli
BS5 with pili. Electron microscope EM 301, Phili...
Figure 1.5 Halobacteria color a saline on the Spanish island Lanzarote red....
Figure 1.6 EHEC O157‐H7 on fibroblast. SEM image, magnification 40,000×.
Figure 1.7
Escherichia coli
cell on a macrophage. SEM image, magnification 4...
Chapter 2
Figure 2.1
Staphylococcus aureus
. SEM image, magnification 25 000×.
Chapter 3
Figure 3.1 Examples of measuring device labels: Green, round calibration sti...
Figure 3.2 Beam balance from earlier times.
Chapter 4
Figure 4.1 Schematic of the passages.
Chapter 5
Figure 5.1 Hygieia fountain in the courtyard of Hamburg City Hall, erected a...
Figure 5.2 Impression of an unwashed hand on a contact plate with culture me...
Figure 5.3 Standardized rub‐in method for hygienic hand disinfection accordi...
Figure 5.4 Putting on hairnet, beard, and mouth guard.
Figure 5.5 Disposable clothing for visitors and maintenance personnel in RRK...
Figure 5.6 Clothing for employees in RRK “B.” The used cleanroom clothing is...
Figure 5.7 Running tracks of a grain weevil (
Sitophilus
sp.) on an agar plat...
Chapter 6
Figure 6.1 Sedimentation plate (CSA). Typical of airborne bacteria is the pr...
Figure 6.2 Lockable contact plates with CSA and four neutralizers. Contact o...
Figure 6.3 Using the example of purified water, the action limit is 100 CFU/...
Figure 6.4 Illustration of the alert and action level.
Figure 6.5 Typical yellow colonies of
Micrococcus luteus
, with positive cata...
Figure 6.6
Micrococcus luteus
in the microscope, magnification 400×, Nikon p...
Figure 6.7
Bacillus cereus
.
Figure 6.8
Bacillus atrophaeus
on CSA. Before renaming, this bacillus specie...
Figure 6.9
Serratia marcescens
on CSA isolated in environmental monitoring, ...
Figure 6.10
Pseudomonas
sp. photographed under UV light.
Figure 6.11 Streptomyces species. Nikon Photomicroscope, magnification 400x....
Chapter 7
Figure 7.1 Schematic of the microbiological quality control of solids.
Figure 7.2 (a) Activation cascade in amoebocytes triggered by endotoxins (fa...
Figure 7.3 Diaporama of horseshoe crabs (models), exhibited at Oceanum, Stra...
Figure 7.4 Gelation reaction in the tube: on the left, gelation = endotoxin‐...
Figure 7.5 Required reagents including human whole blood from a donor. Measu...
Figure 7.6 To check the success of sterilization in the autoclave: autoclave...
Figure 7.7 The bioindicator
Bacillus stearothermophilus
is applied to round ...
Figure 7.8 Formula of gentamycin.
Chapter 8
Figure 8.1 Evaluation workstation according to Ph. Eur. chapter 2.9.20 “Test...
Chapter 9
Figure 9.1 Artificial biofilm with
Pseudoalteromonas ruthenica
on a ceramic p...
Figure 9.2 The inner wall of the stainless steel boiler (volume 5000 l) show...
Figure 9.3 Legionella colonies on GVPC agar.
Figure 9.4 Typical mirror gel colonies of
Legionella pneumophilia
.
Figure 9.5 Membrane filtration (polycarbonate membrane, 0.45 μm). Yellow col...
Figure 9.6
Legionella pneumophila Corby
. SEM image, magnification 40 000×....
Chapter 10
Figure 10.1 Chemical formula of adenosine‐5′‐triphosphate (ATP).
Figure 10.2 Conversion of 5‐(6)‐carboxy‐fluorescein diacetate (CFDA) to 6‐ca...
Figure 10.3 Fluorescent colonies of
Aspergillus brasiliensis
.
Chapter 11
Figure 11.1 Endosafe
®
nexgen‐PTS™.
Chapter 12
Figure 12.1 Fishbone diagram exemplary for the LAL test, created with MS‐Vis...
Figure 12.2 Failure mode and effects analysis (FMEA) exemplary for the LAL t...
Chapter 13
Figure 13.1 Classical light microscope, manufactured by Leitz in 1924, maxim...
Figure 13.2 Laboratory with Electron Microscope EM 301, Philips, Eindhoven, ...
Figure 13.3
Escherichia coli
with flagella. Shadow casting procedure 45° (ar...
Figure 13.4
Clostridium pasteurianum
. Phase contrast, magnification 400x, Ni...
Figure 13.5 Capsule of
Bacillus megaterium
with China Ink. Phase contrast, m...
Chapter 14
Figure 14.1 Sinner's circle.
Chapter 17
Figure 17.1 BfArM in Bonn‐Bad Godesberg, Germany.
Figure 17.2 EDQM in Strasbourg, France.
Figure 17.3 The Paul Ehrlich Institute in Langen (Hesse, Germany).
Figure 17.4 The Robert Koch Institute in Berlin, Germany.
Guide
Cover
Table of Contents
Title Page
Copyright
Preface to the English Edition
Preface to the First German Edition
List of Abbreviations
Glossary
Begin Reading
Literature
Index
End User License Agreement
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Pharmaceutical Microbiology
Best Practices, Validation, Quality Assurance
Michael Rieth
Author
Dr. Michael Rieth
Fiedlerweg 9
64287 Darmstadt
Germany
Cover Image: © VERO/Veronika Emendörfer, Darmstadt www.veronika-emendoerfer.de
All books published by WILEY‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
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A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
© 2025 WILEY‐VCH GmbH, Boschstraße 12, 69469 Weinheim, Germany
The manufacturer's authorized representative according to the EU General Product Safety Regulation is Wiley‐VCH GmbH, Boschstr. 12, 69469 Weinheim, Germany, e‐mail: [email protected].
All rights reserved (including those of translation into other languages, text and data mining and training of artificial technologies or similar technologies). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Print ISBN: 978‐3‐527‐35425‐2
ePDF ISBN: 978‐3‐527‐84870‐6
ePub ISBN: 978‐3‐527‐84869‐0
oBook ISBN: 978‐3‐527‐84871‐3
Preface to the English Edition
For the third edition of “Pharmaceutical Microbiology,” now written in English, the currently topical subject of Bacterial Endotoxin Test with recombinant factors will be included in the chapter on endotoxin/pyrogen detection. Wherever possible, references are made to the new editions of the pharmacopoeias Ph. Eur. 11 and USP. The chapters on media fill and on water are updated. The bibliographies have been continued, literature prior to 2000 has been omitted where possible. Finally, the printing errors have been corrected.
The new cover picture is again designed by the Darmstadt artist Veronika Emendörfer/VERO, to whom I extend my sincere thanks.
Darmstadt, March 2024
Michael Rieth
Preface to the First German Edition
This book describes the tasks and activities of biology laboratory technicians and microbiologists working in the microbiology laboratories of pharmaceutical quality assurance. Emphasis is placed on the methods of quality control testing including their validation, the qualification and calibration of the equipment and measuring instruments required for this purpose, environmental monitoring in pharmaceutical and chemical production, and industrial hygiene. A summary of this kind has been lacking in the German‐language literature to date. Primarily bacteriological methods and procedures are presented, but references to cell culture methods and animal models are also given. Modern techniques, keyword “rapid microbiological methods” are also presented. Shorter analysis times are needed, especially for procedures with long incubation times, such as sterile testing and testing for mycobacteria and mycoplasmas. The future will show whether these methods can permanently replace the classical cultivation techniques, which largely date back to Robert Koch and other bacteriologists of the late nineteenth century.
I would like to thank the following colleagues for providing photos and illustrations: Daniela Grabis (Merck KGaA), Monika Lamoratta (Lanxess Deutschland GmbH, Leverkusen), Peter Hilgendorf (Daiichi Sankyo Europe GmbH, Pfaffenhofen), Matthias Nagel (Bremerhaven University of Applied Sciences), Armin Quentmeier (University of Dortmund) and Manfred Rohde (Helmholtz Centre for Infectious Diseases, Braunschweig). I thank Barbara Gerten (Merck KGaA) for valuable suggestions and literature references.
I would especialy like to thank the artist VERO/Veronika Emendürfer, Atelier Holzhofallee.
List of Abbreviations
AL
Action limit or action level.
AMG
Arzneimittelgesetz.
AMWHV
Arzneimittel‐ und Wirkstoffherstellungsverordnung.
AP
Aqua purificata (purified water).
API
Analytical process index.
atm
Atmosphere.
ATCC
American Type Culture Collection.
ATP
Adenosine‐5′‐triphosphate.
a
w
Water activity.
BfArM
Bundesinstitut für Arzneimittel und Medizinprodukte (Federal Institute for Drugs and Medical Devices).
BG
Berufsgenossenschaft.
BG RCI
Berufsgenossenschaft Rohstoffe und Chemische Industrie (Employer's Liability Insurance Association for Raw Materials and the Chemical Industry).
BiostoffV
Biostoffverordnung.
BLV
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (Federal Office of Consumer Protection and Food Safety).
BMELV
Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (Federal Ministry of Food, Agriculture and Consumer Protection).
BMwA
Bundesamt für wirtschaftliche Angelegenheiten (Austria).
BP
British Pharmacopeia or bubble point.
BPLS
Brilliant green Phenol red Lactose Sucrose Nutrient medium.
BRP
Biological reference preparation.
BSE
Bovine spongiform encephalopathy.
CCIT
Container closure integrity test.
CCOS
Culture Collection of Switzerland.
CDC
Centers for Disease Control and Protection.
CDCP
Centers for Disease Control and Prevention.
CFDA
5‐(6)‐carboxy‐fluorescein diacetate.
CHCA
α‐Cyano‐4‐hydroxycinnamic acid.
cft
Cubic feet.
CIP
Collection de l'Ínstitut Pasteur or cleaning in place.
CLED
Cysteine‐lactose‐electrolyte‐deficient culture medium.
cm
Centimeter.
CPM
Curriculum for Pharmaceutical Microbiology.
CSA
Casein Soymeal Peptone Agar (= TSA).
CSB
Casein Soymeal Peptone Broth (= TSB).
d
Day or diameter.
Da
Dalton, unit of relative molar mass, 1 Da = 1.66018 × 10 exp
−24
g.
DAB
Deutsches Arzneibuch (German Pharmacopoeia).
DAkkS
Deutsche Akkreditierungsstelle.
DAPI
4′,6‐diamidine‐2‐phenylindole.
DEHS
Diethylhexylsebacic acid.
DEV
Deutsches Einheitsverfahren.
DGFM
Deutsche Gesellschaft für Mykologie e.V. (German Society for Mycology).
DGHM
Deutsche Gesellschaft für Hygiene und Mikrobiologie e.V. (German Society for Hygiene and Microbiology).
DIF
Direct immunofluorescence.
DIMDI
Deutsches Institut für Medizinische Dokumentation und Information (German Institute for Medical Documentation and Information).
DIN
Deutsche Industrienorm.
DKD
Deutscher Kalibrierdienst.
DMSO
Dimethyl sulfoxide.
DNA
Desoxyribonucleic acid.
DOP
Dioctyl phthalate.
DQS
Deutsche Gesellschaft zur Zertifizierung von Managementsystemen (German Association for the Certification of Management Systems).
DSM
Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms).
DSMZ
German Collection of Microorganisms and Cell Cultures.
EAME
Federal Office of Metrology (Switzerland).
ECCO
European Culture Collections Organization.
EDQM
European Directorate for the Quality of Medicines and Healthcare.
EDTA
Ethylenediaminetetraacetic acid.
EE
Endotoxin units.
EEU
Endotoxin equivalent unit.
EHEC
Enterohemorrhagic
Escherichia coli
.
ELC
Endotoxin limit concentration.
ELISA
Enzyme‐linked immuno sorbent assay.
EtOH
Ethanol.
EU
Endotoxin unit (1 EU = 1 IU = 1 IE).
FAME
Fatty acid methyl ester.
FDA
Food and Drug Administration.
FLI
Friedrich‐Löffler‐Institut.
GBF
Society for Biotechnological Research.
GenTG
Genetic Engineering Act.
GLP
Good laboratory practice.
GMP
Good manufacturing practice.
GSF
Society for Radiation and Environmental Research.
GVPC
Glycine, vancomycin, polymyxin, cycloheximide agar.
h
Hour.
HEPA
High efficiency particulate airfilter.
HRP
Horseraddish peroxidase.
HUS
Hemolytic uremic syndrome.
HZI
Helmholtz–Zentrum für Infektionskrankheiten GmbH.
IDA
International Depositary Authority.
IfSG
Infektionsschutzgesetz (Infection Protection Act).
IPA
Isopropyl alcohol (2‐propanol).
IPT
In vitro pyrogen test.
IQ
Installation qualification.
ISO
International Standardization Organization.
IU
International units.
ISSA
International Social Security Association.
JP
Japanese Pharmacopoeia.
CFU
Colony forming units.
λ
Lysate sensitivity Lambda.
l
Liter.
LAL
Limulus amoebocyte lysate.
LER
Low endotoxin recovery.
LF
Laminar flow.
LMX
Lauryl sulfate MUG X‐galactopyranoside.
LPS
Lipopolysaccharide.
LRW
LAL reagent water (Ph. Eur.: water for BEP) or limulus reagent water.
LTA
Lipoteichoic acid.
MALDI/TOF
Matrix‐assisted laser desorption/ionization – time of flight.
MAT
Monocyte activation test.
m‐CP
modified Cellobiosis Polymixin B Agar.
min
Minute.
MRI
Max‐Rubner Institute.
MRS
Lactobacillus agar according to de Man, Rogosa and Sharpe.
MVD
Maximum valid dilution.
NAT
Amplification of nucleic acids.
NCTC
National Collection of Type Cultures.
NCYC
National Collection of Yeast Cultures.
NP
Novopyrexal.
OB
Object carrier.
OOC
Out of calibration.
OD
Optical density.
ODC
Ornithine decarboxylase.
ÖKD
Österreichischer Kalibrierdienst.
OF
Oxidation/fermentation.
ONGP
o
‐Nitrophenyl‐
β
‐
D
‐galactopyranoside.
OOE
Out of expectation.
OOL
Out of limit/out of level.
OOS
Out of specification.
OOT
Out of trend.
PAO
Polyalphaolefins.
PBS
Phosphate buffered saline.
PCR
Pseudoselagar or polymerase chain reaction.
PDA
Parenteral Drug Association.
PEI
Paul Ehrlich Institute.
Ph. Eur.
European Pharmacopoeia.
pNA
p
‐Nitroaniline.
POD
Peroxidase.
PPC
Product Positive Control.
PPE
Personal protective equipment.
PVP
Polyvinylpyrrolidone (= Povidone).
PVDF
Polyvinylidene fluoride.
PTB
Federal Physical‐Technical Institute.
RCM
Reinforced clostridial medium.
SEM
Scanning Electron Microscopy.
RIA
Radioimmunoassay.
RKI
Robert–Koch–Institut.
RLU
Relative light unit or relative light units.
RNA
Ribonucleic acid.
rpm
Rotations per minute.
RRK
Clean room class (Reinraumklasse).
s
Second.
SAL
Sterility assurance level.
SARS
Severe acute respiratory syndrome.
SAS
Swiss Accreditation Service.
SDA
Sabouraud dextrose agar.
SIM
Sulfide and indole formation, motility.
SOP
Standard operation procedure.
TAMC
Total aerobic microbial count.
TEM
Transmission electron microscopy.
TierSeuchErV
Animal Disease Pathogen Ordinance.
TMB
Tetramethylbenzidine.
TRBA
Technical Rules Biological Agents.
TSA
Tryptic soybean agar.
TSB
Tryptic soybean bouillon.
TTC
Triphenyltetrazolium chloride.
TÜV
Technischer Überwachungsverein (Technical Inspection Agency, Germany).
TVO
(German) Drinking Water Ordinance.
TYMC
Total yeast and mold count.
U
Unit.
UF
Ultrafiltration.
UPU
World Postal Union.
USP
United States Pharmacopeia.
UVV
Accident prevention regulation.
VAAM
Vereinnigung für Allgemeine und Angewandte Mikrobiologie.
VAH
Verbund Angewandte Hygiene (Association for Applied Hygiene).
VBNC
Viable but non‐culturable.
VE
Demineralized.
VfA
Association of Research‐Based Pharmaceutical Companies e. V.
VHP
Vaporized hydrogen peroxide.
VI
Preventive maintenance.
VRBA
Violet red bile agar.
VRBD
Violet red bile dextrose (crystal violet neutral red bile dextrose).
WFCC
World Federation for Culture Collections.
WfI
Water for injections.
WL
Warning limit/alert limit.
WHO
World Health Organization.
WST
Working standard.
XLD
Xylose lysine deoxycholate agar.
ZEBETZ
Central Office for the Registration and Evaluation of Alternative and Complementary Methods to Animal Experiments.
ZLG
Zentralstelle der Länder für Gesundheitsschutz bei Arzneimitteln und Medizinprodukten (Central Office of the Federal States for Health Protection with regard to Medicinal Products and Medical Devices), Germany.
Glossary
Adaptation
Adaptability of microorganisms to changing environmental conditions.
Adhesion
Attachment of microorganisms to surfaces.
Antisepsis
Inhibition or destruction of infectious agents.
Asepsis
Sterility to prevent infection or contamination through the use of disinfection or sterilization.
Chain of infection
Pathway and mode of transmission of the pathogen from the source of infection to the host.
Commercially sterile
Definition in the food sector. The germ content must not increase by more than two powers of ten after incubation in the sealed packaging for 14 to 21 days. Pathogenic and toxigenic germs must not be detectable per gram.
Contamination
Colonization or adherence of infectious agents to objects.
Decontamination
Extensive elimination of a contamination.
Disinfection
Defaunation, disinfestation, i.e. destruction of harmful small organisms.
Endemic
Persistent, never‐ending state of infestation within the population; endemic diseases include gonorrhea and syphilis.
Epidemic
Temporally clustered occurrence of an infectious disease within a population group, e.g. cholera, typhoid, plague, smallpox.
Epidemiology
The study of the cause and spread of infectious diseases.
Germ carriers
Persons who harbor pathogens without being ill (see also permanent carriers).
Immunity
Acquired resistance to a specific pathogen species or increased reactivity against a specific antigen due to previous contact with it.
Infection
Invasion of pathogens into the host organism with subsequent multiplication and reactions of the host.
Infectious disease
Result of the interaction between the attacks of the pathogen and the defenses of the host.
Incubation period
Time between the entry of a pathogen and the appearance of the first symptoms of disease.
Intrathecal
Within the theca folliculi and the theca medullae spinalis.
Latent infection
Hidden infection without signs of disease.
Lethality
Number of people who died within a certain period of time in relation to the number of people with the disease; indicator of the severity of a disease.
Manifest infection
Infection that appears.
Morbidity
Incidence of disease; number of people suffering from a particular disease in relation to the total population (e.g. in relation to 100,000 people). Incidence: number of new cases per time period. Prevalence: number of people with the disease at a given time.
Mortality
Number of people who died from a certain disease in relation to the total population (e.g. in relation to 100,000 people).
Nosocomial
Hospital‐acquired disease.
Pandemic
Major epidemic (influenza; formerly: plague, cholera) spreading across entire continents.
Pathogenicity
Constant property of a germ species to be able to cause disease symptoms in a specific host.
Permanent carriers
Persons who continue to excrete pathogens (e.g. salmonella) after having overcome an illness.
Prophylaxis
Prevention; prevention of disease.
Pyrogens
From Greek
pyr
= fire, all fever‐producing substances including bacterial endotoxins.
SAL
Sterility assurance level = probability that one living microorganism can be contained in one unit of a product after sterilization. The Ph. Eur. Commission is of the opinion that an item can be considered sterile if the theoretical value of no more than one living microorganism is present in 1 × 10
6
sterilized units; SAL = 10
−6
(see also DIN EN 556).
Sepsis
From Greek. putrefaction, synonym septicemia or blood poisoning.
Source of infection
Humans, animals, water, food or objects from which the infection originates.
Sterile
Free from viable microorganisms.
Vector
Carrier of pathogens; often insects (e.g. see malaria).
Virulence
Degree of pathogenicity of a strain of an infectious germ species.
Zoonotic disease
of animals that can also be transmitted to humans.
1Introduction to Microbiology
1.1 Historical
Only a few years after the first descriptions and isolations of microorganisms by Louis Pasteur, Robert Koch, Gerhard Hansen, and others, the public was already aware of an essential property of these mostly unicellular microorganisms: They are ubiquitously distributed, i.e. everywhere! Also, that they can cause fever in the bodies of humans and animals or cause diseases, partly with death consequences, was already known, likewise already disinfection measures like the use of the agents carbolic acid and iodine. Still in 1906, in the 8th edition of the “Lehrbuch der Botanik für Hochschulen” by Eduard Strasburger, among other names of fission fungi (Schizomycetes), the cyanobacteria are called fission algae [1].
Humans have been taking advantage of the services of microorganisms for thousands of years, but without knowing of their existence for very long. The Sumerians brewed a beer‐like beverage as early as 5000 years ago, and the Assyrians fermented grape juice into wine about 3500 years ago.
The first person to see microorganisms with his own eyes was probably the Dutch draper Antony van Leeuwenhoek (1632–1723). He experimented with homemade single‐lens microscopes, with which he achieved magnifications up to 270× and resolutions down to 1.5 μm. In 1675, he examined an infusion of peppercorns and discovered tiny creatures. He discovered more of these creatures, then called “animalcula,” in dental plaque. Van Leeuwenhoek made drawings of these creatures, which he sent by letter to the Royal Society in London in 1683 [2].
The French chemist Louis Pasteur (1822–1895) made several groundbreaking discoveries in the field of microbiology. He experimentally disproved the primordial hypothesis, explained the nature of fermentation using the examples of alcoholic fermentation and lactic acid fermentation, developed methods for disinfection and sterilization, and introduced procedures for combating infectious diseases by vaccination (e.g. rabies vaccination in 1885).
In 1873, the Norwegian physician Gerhard Hansen (1841–1912) microscopically discovered the causative agent of leprosy, Mycobacterium leprae, as one of the first bacteria to be recognized as a pathogen [3]. To this day, this bacterium cannot be cultured in culture media. Diagnosis is made with the microscope on biopsy material or scrapings of the nasal mucosa. The propagation of these mycobacteria is only successful in the paws of mice and the Armadillo. Pathogen‐specific DNA can be detected using the polymerase chain reaction (PCR).
In 1876, the German physician Robert Koch (1843–1910) proved that microorganisms are the causative agents of infectious diseases, using the anthrax pathogen Bacillus anthracis as an example. He established the following four postulates:
Bacteria must be detectable in the infected organism.
These bacteria must be isolated and brought into pure culture.
Infection with these isolated bacteria will cause the disease again in the healthy organism.
The same infectious agent can be isolated from the host again.
Koch developed culture media, e.g. meat extract broth, which he initially solidified with gelatin and later with agar–agar. Koch's plate‐casting method, still used today in all bacteriological laboratories, goes back to him.
Microorganisms are grouped into two taxonomic domains of their own (Bacteria and Archaea) and thus distinguished from the domain Eukarya (fungi, animals, and plants). Based on the cell structure of microorganisms, they are divided into prokaryotes (Bacteria and Archaea; Greek: bakteria = rod; Greek: archaios = ancient, original) and eukaryotes (fungi, yeasts, algae, and protozoa).
1.2 Importance
Medical microbiology is concerned with the study of pathogens of significance to humans and animals, their habits, and their effects on the human or animal organism; it is thus primarily concerned with obligate pathogens (pathogenic in any case) and facultative pathogens (pathogenic under certain circumstances), i.e. with germs that are to be regarded as dangerous or as “pests” due to cell destruction or the release of toxic metabolic products. However, microorganisms are generally much more likely to be considered “beneficial organisms.” Biological equilibrium without microorganisms is not possible at all. By mineralizing organic matter (e.g. plant material), they ensure the recovery of carbon, nitrogen, sulfur, phosphorus, etc., which are then available again to the plants (material cycles). In the gastrointestinal tract of humans and animals, microorganisms play an important role in the digestion of food. The skin and mucous membranes of humans are also colonized. To illustrate the orders of magnitude: A human being consists of about 1013 cells. The gastrointestinal tract is home to about 1014 and the skin to about 1012 microorganisms, which together weigh about 1.25 kg [4]. The human body thus harbors more microorganisms than it has cells of its own.
Microorganisms find application in the food industry. Examples are:
Yeasts in the manufacture of bread, beer, sake, and wine;
Lactic acid bacteria in the production of yogurt, kefir, sauerkraut, and salami;
Acetic acid bacteria for the preparation of vinegar;
Molds in cheese production (Gorgonzola, Roquefort, etc.) and for the preparation of soybeans (in East Asia).
Microorganisms are used for the recovery of:
Vitamins;
Amino acids;
Hormones;
Steroids;
Enzymes, e.g. amylases (starch cleavage), proteases (digestion, leather tanning), lipases (fat cleavage), and pectinases (fruit juice clarification);
Antibiotics;
Alcohols (ethanol, butanol, butanediol, glycerol, etc.);
Active ingredients, some of which are also produced by genetically modified microorganisms (e.g. insulin).
Microorganisms are essential in the treatment of wastewater and waste composting.
1.3 World of Microorganisms
An overview of the various groups of microorganisms and other causative agents of infectious diseases is given in Table 1.1. Microorganisms are not visible to the naked eye; for their observation, a light microscope is required, and in the case of viruses – with very few exceptions – an even higher magnification electron microscope is required.
Table 1.1 Groups of microorganisms and biological agents.
Subcellular biological objects
Mostly unicellular organisms (microorganisms)
Prions
Prokaryotes
Viroid
Eubacteria
Bacteriophages
Chlamydia
Viruses
Rickettsia Mycoplasma Archaea
Eukaryotes
Fungi, yeasts, algae, and protozoa
The average size of bacteria is between 0.3 and 10 μm. The diameter of cocci, which belong to the human skin flora, is approx. 1 μm. If you think of 500 cocci of this size strung together, the diameter of the dot at the end of the sentence would be reached. Another size comparison: a hair on the head is approx. 40–120 μm, on average 80 μm, thick (see Table 1.2). The human eye can recognize objects up to approx. 25 μm (resolving power).
Table 1.2 Sizes of particles and cells.
Cell or particle
Size
Egg (bird)
In the centimeter range (ostrich egg:
d
= 15 cm)
Ovum (human)
200 μm
Human hair
d
= 40–120 μm, average 80 μm
Human and animal cells
20–30 μm
Human erythrocyte
7.5 μm
Human sperm cell
6.5 μm long
Pollen
7–100 μm
Dust
0.1–100 μm
Aerosols when sneezing
10–300 μm
Protozoa
5–150 μm
Mushrooms
5–10 μm
Bacteria
0.3–10 μm
Nanobacterium equitum
(Archaeon)
0.4 μm
Mycoplasma
0.3–0.8 μm
Chlamydia
0.3–1.0 μm
Rickettsia
0.5–1.0 μm
Viruses
0.016–2.0 μm
Viroid
2 × 40 nm
Macromolecules
1–10 nm
Prions
<
5 nm
Atoms
0.1 nm
d, diameter.
Table 1.3 Living range for molds. In the area marked with X, the growth of molds is optimal.
Temperature (°C)
60
70
75
80
85
90
95
100
pH value
Increase in relative humidity (% r. h.) →
80
12
70
×
×
×
×
×
×
×
11
60
×
×
×
×
×
×
×
10
50
×
×
×
×
×
×
×
9
40
×
×
×
×
×
×
×
8
30
×
×
×
X
X
X
×
7
20
×
×
×
X
X
X
×
6
10
×
×
×
X
X
X
×
5
0
×
×
×
×
×
×
×
4
Increase in the supply of nutrients→.
The world of microorganisms consists of the following groupings (although the following first three groupings are not living organisms in the strict sense, but biological agents).
1.3.1 Prions
Infectious prions PrPsc are misfolded forms of a small (molar mass about 30 000 Da) cellular glycoprotein. The misfolding occurs in cattle between amino acids 121 and 230 and is inaccessible to protease digestion [5]. Stanley Prusiner derived the name from “proteinaceous infectious particle” [6]. PrPsc causes diseases in sheep and goats (scrapie), cattle and cats (bovine spongiform encephalopathy (BSE) and feline spongiform encephalopathy (FSE)), mink, deer, and ungulates. Humans can also be infected (Kuru, Creutzfeldt–Jakob disease, and Gerstmann–Sträussler–Scheinker syndrome). Incubation periods can last for many years. In the course of these diseases, the brain tissue decays in a spongiform manner. BSE first appeared on a larger scale in the United Kindom toward the end of the 1980s, while scrapie has been known for more than 260 years [7]. Presumably, the prions were transmitted via insufficiently heated meat‐and‐bone meal containing PrPsc from scrapie‐infected sheep, which was fed to cattle.
1.3.2 Viroid
Viroids are circular, single‐stranded RNA molecules of low molar mass (approx. 12 × 104 Da, approx. 360 nucleotides). The RNA is “naked,” i.e. not coated by protein. Viroids cause plant diseases, e.g. potato spindle tuber viroid.
1.3.3 Viruses
Viruses (lat. virus = poison, mucus) are predominantly ultramicroscopic, obligate cell parasites that contain only either DNA (e.g. poxvirus and herpes simplex) or RNA (e.g. influenza, rhinitis, and rabies viruses), have no enzyme systems for energy production and no systems for protein synthesis, and cause infected host cells to synthesize the virus building blocks. Viruses consist of at least a nucleic acid‐containing inner body and a protein coat called a capsid. They may be enveloped, i.e. surrounded by a lipid bilayer (such as pathogens of smallpox, herpes, measles, influenza, rabies, acquired immunodeficiency syndrome [AIDS], and severe acute respiratory syndrome [SARS]) or be unenveloped (such as pathogens of polio, hepatitis A, rhinitis, and foot‐and‐mouth disease). Poliovirus can be characterized by the chemical molecular formula C332652H492388N98245 O131196P7501 S2340[8]. On December 9, 1979, the WHO declared the world free of smallpox.
The size of viruses varies from 20 nm (picornaviruses and arboviruses) to 2000 nm (plant viruses such as the Citrus tristeza virus). Viruses that infect bacteria are called bacteriophages. T phages (coli phages) are well studied in molecular biology; their size is 70 nm × 200 nm. The bacteriophage T4 contains linear double‐stranded DNA (dsDNA), and its genome size is 168 903 (see Figure 1.1).
Table 1.4 Environmental conditions for microorganisms.
Parameter
Lower limit
Medium range
Upper limit
Temperature
<−12 °C no growth possible
e.g.
Escherichia coli
: 8–48 °C, optimal 39 °C
113 °C
Pyrolobus
, 84 °C
Bacillus
sp. from approx. 112 °C sporicidal effect in autoclave
pH value
pH 0.5
Picrophilus oshimae
Picrophilus torridus
pH 5.5–8.0
pH 11.5
Bacillus‐like
isolates and pH 13.0
Natronobacterium
—
Isolate MT41 grows optimally at 700 bar and 4 °C
[24]
>
1000 bar (deep sea), isolate MT41
Water activity
a
w
= 0.60
Saccharomyces rouxii
a
w
= 0.80–0.99
a
w
= 1.0 (pure water) water microorganisms
Salt concentration
0.9 % (=0.16 M NaCl)
5 M NaCl (=29% w/v),
a
w
= 0.75
Haloferax volcanii
(see
Figure 1.5
)
Radiation
—
—
18 000 Gy
Deinococcus radiodurans
Figure 1.1 Bacteriophage T4, acceleration voltage 80 kV, uranyl acetate as contrast medium, magnification 184 800×. Electron Microscope EM 301, Philips, Eindhoven, The Netherlands.
In 2003, large viruses were found in amoebae. They were called mimiviruses. With sizes up to 800 nm, they are visible under the light microscope [8]. Viruses are detected using tissue culture, animal experiments, egg culture methods, PCR, and immunological methods. Approximately 1500 viruses are currently known, of which slightly more than 200 are human pathogens [9].
1.3.4 Archaea
Archaea (Greek: archaios for old, original) live in extreme locations, for example, in salt lakes (e.g. Dead Sea with approx. 30% of different salts, corresponding to an aw value of 0.75), hot sulfur springs, and the deep sea. Archaea include methanogenic (produce methane, CH4), thermophilic (live at high ambient temperatures), and halophilic representatives. Their cell wall structure is different from that of bacteria. So far, more than 250 species of archaea have been described, although pathogenic representatives are not yet known [9].
The smallest representative of the archaea is Nanoarchaeum equitans. Although this organism has its own ribosomes, it uses part of the metabolic functions of the host cell. Archaea were defined as a separate bacterial kingdom (domain) by the American microbiologist C.R. Woese in 1977 [10].
1.3.5 Bacteria
Bacteria reproduce asexually by transverse division. They have a rigid cell wall of varying thickness that ensures shape and stability. The nuclear structure (which is not a true nucleus) is called a nucleoid. Meanwhile, more than 1000 bacterial genomes have been sequenced (the first sequence analysis was achieved in 1995 on the genome of Haemophilus influenzae). To date, more than 10 000 bacterial species have been described [11], and several hundred are added each year. Approximately 340 of the species known to date are human pathogens, and among the causes of death, infectious diseases occupy second place, with the consequences of tobacco consumption in the first place [12].
1.3.6 Chlamydia
They are obligate cell parasites that possess all the typical structural elements of bacteria. Chlamydiae undergo a developmental cycle (from the 0.3 μm elementary bodies to the 1 μm initial bodies). An example is the causative agent of the parrot disease psittacosis, Chlamydia psittaci, which can also infect humans, developing flu‐like symptoms. Infection happens through inhalation of dust containing chlamydia from bird excrement. Many pigeons in cities are infected with Chlamydia species.
1.3.7 Rickettsia
They are also obligate cell parasites of 0.5–1 μm size. They reproduce by transverse division with the aid of host cell cofactors. An example is the causative agent of spotted fever, Rickettsia prowazekii. The bacteria are transmitted by ticks, mites, lice, and fleas. Another pathogen is Coxiella burnetii. Domestic and wild animals are infected by tick bites. Humans become infected by dust containing Coxiella from animal feces. The disease is called Q fever, and its diagnosis is done serologically.
1.3.8 Mycoplasma
This group includes bacteria without a rigid cell wall; as a result, they appear polymorphic and show high plasticity. Their size is 0.3–0.8 μm. Examples are the pathogens of pneumonia (Mycoplasma pneumoniae) and urinary tract infections (Ureaplasma urealyticum). Normal flora include Mycoplasma buccale (on the oral mucosa) and Mycoplasma hominis (on the mucosa of the intestine). From Mycoplasma genitalium, the genome was sequenced. It is 580 kb in size and contains only about 500 genes. In the Gram stain, the mycoplasmas react variably. They are resistant to penicillins and sulfonamides but not to tetracyclines and streptomycin.
1.3.9 Fungi
Fungi (mycobionta, molds, and yeasts) are a very heterogeneous group of ubiquitous eukaryotic organisms in many forms and colors, with more than 110 000 species. They can be divided into the following four groups: Basidiomycota with about 30 000 species, Ascomycota with about 46 000 species (including about 1000 species of yeasts or Endomycetes), Zygomycota with about 650 species, and Fungi imperfecti (or Deuteromycota) with about 30 000 species. Almost all human and animal pathogenic fungi, as well as most molds, belong to this last group [13]. Fungi are estimated to make up 25% of the biomass of our planet. Fungi can even colonize optical lenses in objective lenses. About 300 species of fungi are pathogenic to humans [9], and most diseases of crops are due to fungi. Fungi can produce toxins (more than 500 mycotoxins are known to date), some of which are lethal to humans and animals (in Germany, about 50 people die annually as a result of fungal poisoning). In addition, toxic and carcinogenic metabolites can be produced, especially by molds (e.g. Aflatoxins, Ochratoxins, Patulin, and Fusarium toxins). The Food and Agricultural Organization estimates that up to a quarter of the world's food production is contaminated with mycotoxins. The allergenic potential of the fungi, on the other hand, has so far been classified as low.
Common to all fungi is a rigid cell wall containing chitin (a polysaccharide), cellulose, glucans, etc., and the true nucleus. Fungi cannot photosynthesize, so they feed on finished organic matter: they are C‐heterotrophs. Fungi either feed on dead organic matter (see Figure 1.2) or live as parasites on or in other living organisms. While most of the fungi reproduce asexually, some fungi also reproduce sexually. Fungi imperfecti are only known to reproduce asexually, for example, by sprouting or conidiospores. Fungi are unicellular (e.g. sprout fungi such as the brewer's yeast Saccharomyces cerevisiae and the various Candida species) or multicellular (e.g. pathogens of dermatomycoses). The fungal cells are significantly larger than bacterial cells. Sprout fungi can appear in severely ill patients, for example, on the tongue, in the throat, in the bronchi, and in the esophagus. They also cause dangerous diseases of the meninges, lungs, kidneys, intestines, and other organs. Aspergillosis, caused by Aspergillus fumigatus, is feared in hospitals: this infectious disease has the worst prognosis of all [14]. A photograph of an Aspergillus is shown on Figure 1.3. In nature, the fungus lives on dead plants, in compost heaps, organic waste garbage cans, cereals, hay, tea leaves, and nuts. The living range for molds is shown in Table 1.3. The fungal spores are inhaled through the lungs. In healthy people, the spores are destroyed by macrophages, but in immunocompromised patients, the defense against fungal spores does not work, and they are transported through the bloodstream to the various organs. The lethality is high; about 2/3 of the infected patients die, which is about 2500 people in Germany every year.
Table 1.5 Minimum water activity values (aw) for the growth of various microorganisms. Below 0.60, growth is no longer possible.
Source: Compilation from Refs. [18–21]
a
w
value
Microorganisms
Substrate
Representative
0.98–1.00
Waterborne bacteria
Pure water 1.0; blood, parenterals, nasal spray, and hair shampoo 0.99
Caulobacter and Spirillum
0.96–0.97
Gram‐negative rods
Juices, creams; 7.5% w/v NaCl solution 0.957
Pseudomonas,
Escherichia coli
, Shigella, Acinetobacter, Flavobacterium, and many other microorganisms
0.91–0.95
Most bacteria
Bread, ham 0.89–0.96
Bacillus cereus
, Clostridium, Citrobacter, Corynebacterium, Salmonella, Lactobacillus, and Serratia
0.87–0.94
Most yeasts
Maple syrup, jam, and liquid oral formulations
Candida, Fusarium, Mucor, and Aspergillus
0.86–0.90
Gram‐positive cocci
Salami
Micrococcus and
Staphylococcus aureus
0.80
Most molds
Cake; jam
Penicillium, Rhizopus, and
Saccharomyces bailii
0.70
Molds
Cereals
Aspergillus glaucus
0.75
Halophilic bacteria
Salt lakes; salted fish
Halobacterium and Halococcus
0.65
Xerophilic molds
Cereals; cookies; dried fruits
Aspergillus, Chrysosporium, Xeromyces, and Eurotium
0.61
Osmophilic yeasts
Ointments 0.55
Zygosaccharomyces rouxii
and
Xeromyces bisporus
Figure 1.2 White mold on damp furniture wood in the basement, after rainwater penetration.
Skin fungi belong to various species and, like sprout fungi, are very difficult to control. Fungi can multiply in damp places, for example, in bathing establishments.
Other fungal diseases are liver tumors caused by fungal metabolites (aflatoxins and patulin). Aflatoxin can be present in moldy food, patulin‐containing spoiled apples, and juices.
As with foodstuffs, mold growth is also possible with pharmaceuticals, especially if they are stored improperly. Walls with mold growth pose a particular hazard, as measurably elevated levels of fungal spores can be found in the air in such rooms. This is a danger both for the people who have to stay in such rooms and for the medicines that are manufactured or stored in such rooms.
1.3.10 Protozoa
This group includes free‐living or parasitic unicellular eukaryotes with most of the characteristics of animal cells. Reproduction usually occurs by bipartition. Transmission of parasitic protozoa to humans often occurs through arthropods: The causative agent of malaria (Plasmodium) is transmitted by anopheles mosquitoes, and the causative agent of sleeping sickness (Trypanosoma brucei) is transmitted by tsetse flies (Glossina ssp.). Sleeping sickness is one of the few infectious diseases with a 100% lethality rate.
1.4 The Bacterial Cell
The average weight of a bacterial cell is about 10−12 g, which is less than one‐thousandth of the cell weight of an animal cell [15], and it is also much smaller than the eukaryote cell. The bacterial cell is composed of the following components:
Prokaryotic nuclear substance (nucleoid)
The nucleoid is a naked, unraveled, right‐handed, mostly circular DNA molecule with a molar mass of about 2.5 × 10
9
Da. In case of transverse division, doubling of the nucleoid always occurs first.
Plasmids
Plasmids consist of extrachromosomal DNA. Between 1% and 5% of the genetic information of the bacterial cell may be plasmid‐encoded. Of medical importance are the resistance plasmids (R plasmids), which contain genes that provide resistance to antibiotics. The F plasmids carry fertility factors.
Cytoplasm
The cytoplasm contains many substances dissolved in water (proteins and minerals) and the 70S ribosomes. The ribosomes are responsible for protein synthesis. Their number in fast‐growing bacteria is about 20 000, their size is 20–24 nm, and their sedimentation speed in ultracentrifuge is 70 Svedberg units.
Reserves
Reserve substances include polyphosphates (volutin), poly‐β‐hydroxy‐butyric acid (PHB), glycogen (in
Bacillus species
and enterobacteria), and lipid droplets.
Reserve substances are formed under certain environmental conditions and are used again in situations of deficiency.
Cytoplasmic membrane
This semipermeable elemental membrane consists of a phospholipid bilayer in which folded protein molecules are embedded.
Cell wall
It is 10–80 nm thick, gives the bacteria a solid shape, and forms an elastic protective shell against external injuries. The internal pressure can be between 500 and 2000 kPa
[9]
. The cell wall is permeable, i.e. largely permeable to food substances. The chemical structure of the cell wall is different in Gram‐negative and Gram‐positive bacteria. In Gram‐positive bacteria, the cell wall consists of a lot of murein (mucopolysaccharide cross‐linked by peptides). The thickness of the cell wall is 15–80 nm. The cell wall makes up 30% of the dry mass. In Gram‐negative bacteria, there is little murein but many proteins and phospholipids. The thickness here is around 10 nm.
1.4.1 Capsule
Many bacteria form a capsule of polysaccharide polymer outside the cell in vivo with the aid of extracellular enzymes (except Bacillus anthracis: D-GLUTAMIC ACID). The capsule largely protects against phagocytosis (uptake by white blood cells) and thus against nonspecific infection defense.
1.4.2 Flagella
Most motile bacteria have flagella. These are made up of the linear protein flagellin. Flagella are anchored in the cell envelope via a complex structure and are able to rotate around their axis (at frequencies of up to 300 Hz), resulting in forward movement. In water, bacteria can thus advance at up to 100 μm s−1. Escherichia coli has four to six flagella (see Figure 13.3), whose lengths can be up to 45 μm [16]. Flagella of bacteria are thin (15–20 nm), and archaeal flagella are only 10–13 nm in width [20].
The flagella can be arranged as follows:
Monotrich (e.g.
Vibrio
)
Lophotrich (e.g.
Pseudomonas
)
Peritrich (e.g.
Salmonella
)
1.4.3 Fimbriae and Pili
Many bacteria form surface structures that are shorter and finer than flagella. Fimbriae are responsible for attaching to specific host cell receptors. Sex pili are filamentous hollow protein tubes responsible for cell‐to‐cell contact during conjugation (transfer of DNA). The pili (see Figure 1.4) are 0.2–1.2 μm long and 10 nm thick [16].
Table 1.6 Human‐pathogenic Escherichia coli strains.
Pathovar
Property
Disease
Adherent
Escherichia coli
EAEC
Aggregation
Role of DAEC in intestinal
DAEC
Adherence
Infections unclear
STEC/VTEC
EHEC
Adherence
Hemorrhagic colitis, HUS
EIEC
Invasion
Dysentery‐like colon infection
EPEC
Adherence
Diarrhea in babies
ETEC
Colonization
Traveler's diarrhea
DAEC, diffusely adherent Escherichia coli; EAEC, enteroaggregative Escherichia coli; EHEC: enterohemorrhagic Escherichia coli; EIEC, enteroinvasive Escherichia coli; EPEC, enteropathogenic Escherichia coli; ETEC, enterotoxigenic Escherichia coli; STEC, Shiga toxin‐producing Escherichia coli; VTEC, verotoxin‐producing Escherichia coli; HUS: hemolytic uremic syndrome.
Figure 1.3 SEM image of Aspergillus niger on agar plate.
Source: Courtesy of Dr. M.Rohde/HZI Braunschweig.
Figure 1.4Escherichia coli BS5 with pili. Electron microscope EM 301, Philips, Eindhoven, The Netherlands.
1.4.4 Endotoxins
Endotoxins are lipopolysaccharides (LPS) localized in the outer membrane of the cell wall of Gram‐negative bacteria. They enter the milieu through the release of membrane vesicles by living bacteria or when the bacterial cell dies. Endotoxins have a fever‐producing (pyrogenic) effect in humans and in many mammals (rabbits, dogs, cats, horses, cows, etc.), but not, for example, in birds.
1.4.5 Exotoxins
The Exotoxins are microbial virulence factors. As the prefix “exo” indicates, these toxins are secreted into the environment or are associated with the outside of the microbial cell. Exotoxins are mostly proteins/enzymes. Vibrio cholera is an example of a bacterium that contains endotoxins and exotoxins. Differential characteristics of exotoxins and endotoxins are shown in the following table.
Characteristic
Exotoxin
Endotoxin
Chemical nature
Protein
Lipopolysaccharide
Part of Gram(−) cell
No
Yes
Most from Gram(+) bacteria
Yes
No
Usually extracellular
Yes
No
Phage or plasmid coded
Many
No
Antigenic
Yes
Weakly
Can be inverted to toxoid
Many
No
Neutralized by antibody
Yes
Weakly
Differing pharmacologic specificities
Yes
No
Stable to boiling
No
a
Yes
Exception is the enterotoxin of Staphylococcus aureus; the enterotoxins withstand boiling.
Source: Reference [34]/with permission of McGraw‐Hill.
1.4.6 Bacterial Morphology
The size range of bacteria and other microorganisms is shown in Table 1.2. The size of all living organisms ranges between 0.3 μm (smallest bacteria such as Corynebacterium diphtheriae, the causative agent of diphtheria, or Brevundimonas diminuta, a rod‐shaped aquatic bacterium) and the Hallimasch fungus (Armillaria ostoyae), whose mycelial extent under the ground is 600 ha, discovered in 1992 in the US state of Washington [6].
Table 1.7 Infectious diseases and their modes of transmission, pathogens (causative agents), and infectious doses.
Disease
Incubation period
Initiating agent/infectious dose
Transmission
Tuberculosis
4–6 weeks
Mycobacterium tuberculosis
1 cell (guinea pig model)
Aerogenic (oral)
Legionellosis
2–10 days
1 cell of
Legionella pneumophila
in respirable aerosol droplets.
Aerogenic
Mite spotted fever
?
3 cells of
Orientia tsutsugamushi
Bite
Q Fever
14–21 days
10 cells of
Coxiella burnetii
Aerogenic
Tularemia
4 days
10 cells of
Francisella tularensis
Aerogenic
Rubella
12–21 days
≥10 Rubella virus (portal of entry pharynx). 60 Rubella virus (entry nasal mucosa)
Aerogenic
Trichinosis
5–10 days
50–70
Trichinella spiralis
Oral
Syphilis
14–28 days
60 cells of
Treponema pallidum
Mucosa
EHEC infection
2–10 days
10–100 enterohaemorrhagic
Escherichia coli
Oral
Flu
1–3 days
340 influenza viruses
Aerogenic
Shigellose/Ruhr
2–7 days
10–200 cells of
Shigella flexneri
10
9
cells of
Shigella dysenteriae
Oral
Campylobacter
3–5 days
500
Campylobacter jejuni
Oral
Enteritis
2 weeks
10
3
Giardia lamblia
Oral
Pulmonary anthrax
1–7 days
≥1300 cells of
Bacillus anthracis
aerogenic
Typhoid
12–14 days
10
5
cells of
Salmonella typhi
Oral
Cholera
1–2 days
>
10
6
Vibrio cholerae
Oral
Food poisoning
4–6 hours 1–3 days
(a)
Bacillus cereus
: oral 10
5
–10
6
Bacilli/g food. b)
Clostridium botulinum
: lethal dose: 0.1–1 μg toxin A
Oral
Diarrhea
Hours
10
8
enterotoxigenic
Escherichia coli (ETEC)
Oral
BSE, scrapie
Years
>
10
5
infectious prions PrP
sc
Oral
Fever
After 20 minutes
Endotoxins of Gram−negative bacteria 1 ng = 0.1 EU from 5 EU/kg body weight fever reaction
Intravenously Intrathecal
Figure 1.5 Halobacteria color a saline on the Spanish island Lanzarote red.
Source: With permission from Dr. Armin Quentmeier.
Table 1.8 Some human diseases and the probable animal sources of infection.
Source: References [9, 32, 33].
Animal source of infection
Disease
Pathogen
Fox, dog, and cat
Echinococcosis
Echinococcus multilocularis
Domestic cats and animals for slaughter
Toxoplasmosis
Toxoplasma gondii
