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Volume 11 No 1 March 1990

Bacterial protein toxins of clinical significance Joseph Alouf, Phd,DSc Head of Bacterial Antigens Unit, Pasteur Institute, Paris, France,

Robert Koch-the father of medical bacteriology Eric Bridson,CBiol,FIBiol ,FIMLS Technical Consultant , Oxoid Ltd.

An enlightening account of the life and achievements of one of the pioneers in the field of medical

A review of the most important recent developments in the field of protein and peptide toxins. "--"'''---~_=d.>~ bacteri ology.

Bacterial protein toxins of clinical significance Joseph E Alouf, PhD, DSc, Professor of Immunochemistry, Head of Bacterial Antigens Unit, Pasteur Institute, Paris, France.

The aim of this review IS to scan some of the most Important achievements in the field of protein and peptide toxins In the past decade and to provide current Information on newly discovered toxins of clinical significance.

Bacterial loxmorogy , the biological discipline dealing with the study of bacterial toxins, IS presently one of the mosl Important areas of clinical microbiology. This discipline has attracted in the past two decades a great number of Investigators from various fields: biophysIcs and biochemistry, molecular microbiology, genetics. Immunology. cell and molecular biology, neurophysiology and pathophysiology. A precise definition of bac­tenal toxins IS a difficult task.

1 These agents

can be defined operationally as a collecllon of simple or complex proteins and peptldes (occasionally associated with carbohydrate or lipid mOieties) and Ilpopolysaccharides of bacterial orig in, which Interact direct ly wittl molecular ta rgets and cause small amounts of cellular damage andlor Impairment of one or several physiological functions of a human. animal or plant organism. This damage may lead to overt disease and sometimes host death.

This definition IS. however. limited and it eliminates from the field of bacterial toxins: (i) the tOXIC non-peptide low molecular weight molecules produced by bactena. (ii) the macromolecular bacterial substances which are non-toxIC by themselves (such as tuber· culin and mallein) but activate the Immune system by mechanisms leading to hyper­sensitivity or ImmunopathologICal damage. (iii) bacterial enzymes and other proteins which are not' tOXIC by themselves (such as hyaturomdases and prot eases) but may contribute as virulence lactors to assist the tOXIC effects of a toxin.

The lipopolysaccharide toxins are the so-called bactenal endotoxlns which playa major role In the pathogenesIs of endotOXin­Induced shock In patients severeJy Infected by Gram·negatlve bactena. These toxins are st ructural components of the external layer of Il1e outer membrane 01 these bacteria. Endo­toxins exhibit Impressive pharmacological. Immunological and tOXIC activities which have been the subject of Innumerable Investlgallons."

Protein and peptide toxins are produced by both Gram-posilive and Gram-negative bactena. They fall Into two broad classes: (I) exoloxlns which are released by these bacteria In ttle surrounding enVIronment after crossing the cell envelopes during act ive growlll or toward the end of the growth cycle: (II) cell­bound-toxins which remain In the bactenal cytoplasm or pen plasm or associate 10 cell membrane. Such toxins may be released In the surrounding enVIronment upon cell lysIs. About 70% of protein and peptide toxins are exotoxlns

Figure, : Mouse peritoneal macrophages treated with the membrane damaging toxin streptolysin o from group A streptococci.

Progress in the study of bacterial protein and peptide toxins The first bacterial protein toxin d iscovered was diphtheria toxin whict1 was characterised In 1888 by Emile Raux and Alexandre Yersin at the Pasteur Institute In Pans as a poisonous substance precipitated from a broth culture of diphtheria bacilli. The two other major. 'claSSIc' tOXins. te tanus and bo tulrnum. were discovered in 1890 and 1896 respect ively by • Kunl. Faber 8riedel and Fraenkel lor the former and by van Ermengem for the latter Since then about 240 protein and peptide toxins l1ave been described In the scientifiC literature up to 1989 (Table 1' .

More than 100 toxins have been punfled to homogeneity dunng the past decade and conSiderable data regarding molecular weight. sub-unit structure and amino aC id sequences are avatlable.3. ~ More tl1an 60 toxin structural genes have been c loned and tllelr nucleotide

sequence established. Thirteen toxins l1ave al ready been crystallized and the three­dimenSional structure of one of them . Pseudomonas aerugmosa exotoxin A has been determined at 3A resoJulton .5

Tl1e mechanism of achon at tl1e cellular and molecular levels of many claSSical and newly discovered toxins IS presently known In great detail. ThiS progress In bacterial toxlnology can be attributed to the four major approaches WhlC~l have dOminated biology in the past two decades: (i) gene manipulation tecllnology. (ii) modern cell biology including. transmembrane­signalling systems and the mechanisms of endocytOSIS by eukaryollc cells. (iii) monoclonal antibodies and the development of cellular Immunology. and (Iv) the new technologies of protein separation. The state of the art of the development of toxlnologlcal research dUring tl1 e past fifteen years has been covered in many books and reviews. 1 ~ 6 , 8

Table 1: Bacterial toxins repertoire (proteins and peptides)

Total number known: 238 (June 1989) 115 (48%) produced by Gram + ve bacteria 123 (52%) produced by Gram - ve bactena Extracellular : 75% Intracellular: 25%

Membrane-damaging (cytolytic) toxins: 90 (37%) 01 toxin repertoire

52 (Gram + ve bacteria) 38 (Gram -ve bactena) Phospholipase activity: 12 toxins (7 Irom Gram + ve bacteria. 5 from Gram -ve bacteria)

Table 2: General mechanisms of action of toxins on target cells

1. Physical damage or disruplion of biomembranes (phospholipases. pore-forming toxins , lipid-binding toxins)

2. Interference with protein synthesis (ADP-ribosylation of elongation factor 2 by diphtheria toxin and P. aeruginosa exotoxin A; ribosomal inactivation by shiga and shiga-like lox ins)

3. Interference wilh intraceJJular regulatory functions (toxins activating G protein subunits: pertussis. cholera and cholera-like toxins; B. pertussis and B. anlhracis adenyl ale cyclases .. .. )

4 . InterferenCe with cytoskeleton functions (actin-depolymeriz ing toxins: C. bOtulinum C2 and C3 toxins. C. perfringens iota toxin , C. spiroforme toxin)

5. Interference with neuronal functions (inhibition of neuromediator transmission by tetanus and botu linum toxins)

6. Interaction with immune system effectors (induction of cytokines and inflammatory cascade mediators by various toxins: e.g .. erythrogenic toxins and toxin-shock syndrome toxin)

7. Antagon isation 01 pharmacological responses at receptor level (blockade of (3 adrenoreceptors by plague lOx in)

Table 3: New membrane-damaging toxins from Gram-positive and Gram-negative bacteria

Toxin

Streptolysin S-li ke cytolysins (1981)

Streptolysin O-like cylolysins 1987. 1989

Co-cytolysin B (CAMP lactor) 1985

S. aureus J·toxin-like toxins (1984, 1987)

Cereolysin AB (1989)

E. coli-like o:-hemolysins (1987)

Hemolytic cytotoxin (1987)

Cell-bound hemolysin (1987)

Hemolysins (15.5 K, 27 K) (1986)

Hemolysin (1987) and protease (1989)

Cytolytic cholera-like toxin (1987) and aeroiysin (1981)

Aerolysin (1988)

Leucotoxin (115 K) (1987)

Hemolysins i and II (1986)

Several cylolysins

Hemolysin (1987)

Hemolytic cytotoxic protease (1989)

General mechanisms of toxicity of protein and peptide toxins In contrast to lipopolysaccharide endotoxins which have a similar structure and mode of act ion. Hle protein and peptide tOxins are extremely diverse In structure and mechan­Isms of tOXICity . These mechanisms may be broadly classified Into seven general types of act ion (Table 2).

Membrane-damaging toxins Almost 40% of protein and pept ide loxlns belong to Ihls category of tox ins also called cytolytic toxins or cytolyslns. Tllese constitute a heterogeneous collection of proteins or peptldes produced by a wide range of Gram­positive and Gram-negative bacteria. ' 9. 10.1 1

TIle vast malorlty of them are haemolytic and are therefore often named haemolyslns. The common feature of these tox ins IS that they damage cell membranes by d isruption andlor disorganisat ion of the bilayer structures which const itute the cytoplasmic membrane of eukaryotlc cells and Ihat of the similar membranes surrounding Intracellular organelles.

Bacteria

Streptococcus agalactiae & S. mutans Treponema hyodysenteriae & T. innocens

Listeria monocytogenes, L. ivanovii, L. seetigeri

Streptococcus agalactiae

Staphylococcus haemolyticus & S. epidermidis

Bacillus cereus

Proteus vulgaris & P. mirabilis, Morganella morganii

Proteus penneri

Serratia marcescens

Klebsiella pneumoniae

Pseudomonas cepacia

Aeromonas hydrophila

Aeromonas sobria

Actinobacillus actinomycetemcomitans

Vibrio cholerae

Other Vibrio spp. (Table 4)

Pseudomonas cepacia

Legionella pneumophila

(I) cleavage of membrane phosphol ipids by a number of loxins exhibiting phospholipase C

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activity such as Clostridium perfringens a-toxin and Staphylococcus aureus .B-toxin, (ii) inter· action of surface-active amphiphilic toxins lead· ing to detergent·like solubi!isation of membrane components such as Bacillus subtilis surlactin, (iii) insertion of peptide or protein domains in the bilayer which generates transmembrane channels or pores as shown for Esc/Jerichia coli a-hemolysin, S. aureus 0' and {j toxins and aerolysin from Aeromonas hydrophila. (iv) modification of membrane stability by toxin interaction, with structural components leading to the formation of complexes as shown for streptolYSin 0 and the related sulfhydryl­activated toxins (Figures 1 and 2).

More than 20 new membrane-damaging toxins, have been characterised In the present decade (Table 3). Many of these are p roduced by bacteria of clinical significance. Various Vibno species other than Vibrio cholerae have been shown In the past five years to produce cytolytic toxins (Table 4). Most of these bacteria were found to be associated with various Infections in humans (wounds. septicaemias. enterotoxic syn­dromes) .12 Among the newly discovered membrane· damaging toxins of clinical signifi. cance IS listeriolysln O. a sulfhydryl-activated toxin (similar to streptolysin 0 and related tOXins), produced by L,stena m ono­cytogenes.13 This toxin appears as a major VIrulence factor implicated in the process of Intracellular growth of this pathogen.

New enterotoxins and other toxins from Gram-negat ive species The enterotoxlns from Gram-negative bacteria characterised by the end of the 1970s were limited to a few which were essentially (i) V. cl70lerae enterotoxin. (ii) E. coli heat labile toxin known as LT-l which is functionally and structurally related to cholera toxin, (iii) E. coli heat·stable tOxin known as ST-1 and (iv) Shiga toxin. Since then a great number of entero­toxins in addition to a variety of other toxins have been isolated. E. coli ST-1 like toxins were found to be produced by Enterobacter cloacae. Klebsiella pneumonrae, Salmonella fyphimurium and other Salmonella species. V .

. cholerae non·0-1 and Citrobacter freundii. Cholera-like cytotoxic enterotoxins were also

Four general mechanisms of damage by cytolyt ic toxins have been cllaracteri sed:

Figure 2: Lysis of human erythrocytes by Streptolysin 0 produced by Streptococcus pyogenes . left: Control. Cent re: 0.5 haemolytic unit of toxin. Right: 5 haemolytic units of toxin.

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Tabl e 4 : New to xins from Vibrio species other than Vibrio cholerae

V. vulnilicus

V. fluvialis

V. mimicus

V. damsela

V. metschnikovii

V. hol/isae

Cytolysin (56 K) , 1985, hemolysin , 1987, dermonecrotic elastase (50 K) , 1967

Cytotoxin (cell·killing·factor 12 K), 1988

Enterotox.in identical to cholera tOxin , 1984

Cytolysin (phospholipase D, 69 K) , 1985

Cytolysin (50 K) , 1988

Enterotoxin (33 K) , 1987

V. parahaemolyticus Thermostable hemolysin (cardiotoxin, en tero·toxin 21 K) , 1973. new related hemolysin , 1988

Table 5 : New toxins produced by E. coli and Shigella s pp

E. coli: Heal·stable enteroloxin II (1918) Heal·labile enterotoxin II (1986) Shiga·like toxin I (verotoxin I) (1983) Shiga·like toxin II (verotoxin II) (1985) Heat·labile cylolethal distending toxin (1988) Cytotoxic necrotising facior (1988)

Shigella spp: Shiga toxin (1903· 1953· 1980) Cytolethat distending toxin (1987)

New non-cytolytic toxins from Gram· positive species

shown \0 be produced by S. lyphl, and S. Iyphllnunum. E. cloacae and K. pneumonlae. Vanous Campy/abacter spp. also produce a SImilar enterotoxin In addition to a cytotoxin and a heat·lablle cytolethal distending toxin. e. pylon (recently renamed Helicobacler pylon) cytotoxin. discovered In 1988, may be relevant to the gastnc ulcer eliCited by thiS microorganism. A great array of new toxins were shown to be produced by E. col! (Table 5). The LT·II, ST·II and shlga·like II tox ins produced by \IllS orgailism are structurally dllferent but functionally SImilar to LT·l, ST·' enterotoxlllS and shlga·llke toxin. A cytolethal distending toxin produced by Shigella spp. was reported III 1989. E. col! st rains which produce cytotoxIc necrotlslng factor'4 onglnally detected In cases of Infant enteritis were subsequently reported In human exira· Intestinal Infections. Another group of toxins from Gram·negatlve bactena IS the family of hepatotoxIc peplldes known as ffilcrocystlns or cyanoglnosrns produced by cyano· bactena.'~

Toxills exhibiting calmodulin·dependent adenyJate cyclase activities produced by Borde/ella appear to be related to the pathogenicity of these organisms.'6 These tox ins share three short homologous regions with Bacillus anthr8cis adenylate cyclase, known as the oedema factor which IS one of the three proteins constituting anthrax toxin. Botulinal neurotoxins type E, F and G were shown to be produced by certain strams of Clostndlum baralJ, C butyncum and C. argentmense respecllvely. Infant botulism was shown to be most often associated with e. botullflum types E and F.

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Clostridium diff,crle 10XIIlS A and Bare major toxins of great clinical significance discovered dUffllg the past decade." In spite of great progress made so far. much work remains \0 be done on these tOXIIlS. Another toxIn of great importance In the pathogeniCity of staphylococcI IS Staphylococcal Toxin· Shock Syndrome toxin 1 discovered In 1981, •

Media for Food Mycology Oxoid offer a comprehenSive range of media for food mycology. There IS an increased awareness of the need for specific media for the isolation and enumeralton of food borne fung I. The Oxoid range of media Include general purpose media such as Rose Bengal Chloramphenicol Agar and Oxyte1racycline GYE Agar, DG18 Agar fo r xerophlhc moulds and AFPA Agar for the isolatron of toxigeniC fungI.

Further Information and details are provided in an OXOId booklet describing media for food mycology.

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and Intensively studied dunng the past five years III many laboratories. This loxln, which IS a potent polyclonal mitogen for human T lymphocytes, was recently shown to affect the release of a number of mediators of the Immune system such as Interferon gamma, Interleuklns·l. ·2. ·3 and tumour necrosis factors alpha and bela 18. 19

Conclusion ThiS short survey has permitted only a glimpse Inlo the development of bactenaJ toxin research over the past ten years. The great achievements In thiS freld are directly connected to clinical microbiology and to the study of bacterial pathogelllcity.

Relerences

Stephen. J and Pietrowski, R A (1986) Bacterial TOXlflS. Van Nostrand Remhold. Wokulgham. UK

2 SlentlvanYI, A el al (1986) Immunoblology and Immunopalliology of B.1Clorral Endotoxlns. Plenum Press. New York

3 Alouf. J E (1988) Bull InS! Pasteur. 86: 127 - 1401 01 Alaul. J E (1990) In Baclerrologle Mf:dlcale (Veron

and L Le mlnOI . EdS). Flammanon PariS 5 Allured. V S el al (1986) Proc Natl Acad Sci

(USA). 83: 1320 - 1324 6 Hardegree. MC ardTu. AT (Eds)(I988)8c1Ctefial

TOXinS. Marcol Dekker . New York 7 Dorner, F and Dtows, J (Eds) (t 988)

Pl1armacoiogy 01 BactOflaJ ToxlIls, Pe1gamon Press, Oxford

8 Harshman. S (Ed) (1988) MICrobial Toxms Tools In Enzymology Meth Enzymol, vot 165. AcademIC Press. New York

9 Freer. J Hand ArbuthnOlt (1976) In Mecl18Il1Sfns m Bac/eoal ToxICology. (Bernhelmer , A W . ed). J Wiley, New YOlk

10 Aloul J E (1977) In The SpeoflClly and Action 01 Anrmal. Bacterial and Plant TOXinS (Cu81recasas, P , Ed ), Chapman and Hall. London

11 Bernl1ellllCt. A.w and Rudy. B. (1986) Blocllom BloptlYS Acla , 864: 123 - 141

12 Kreger. AS (19801) l/lfecllmmun . 14 :326 - 331 t3 Serche P el al (1987) J Immunol, 139:

3813 - 3821 101 De RyckC. J 01 al (1989) J Clm MlcrOVloi. 27:

983 - 988 15 CarmIChael. W W (1989) In Nalural TOXIIlS (Ownby,

C L and Odell. G . Eds). PP 1 - 16, Pergamon Pless. Oxford

16 Masure. H R al al (1989) Mlcroblol Rev . 51: 60 - 65

17 Lyerly. D M el al. (1988) Clm Microbial Rev 1: 1- 18

18 Jupin. C Setal (1988) J Exp Moo . 167: 752-761

t9 Galelh. el at (1989) J Immunot. 142: 2855- 2863

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I I I

Robert Koch the father of medical bacteriology Eric Bridson, CBiol, FIBiol, FIMLS, Technical Consultant, Oxoid Limited , Basingstoke, Hants, UK.

Robert Koch was an extraordinary man who, m ! 873 aI/he age of 30 years. began his research career !O establish Ule role of bacteria In InfectiOuS diseases. By the end 01 the century Koch had risen to the pinnacle of scientific achievement with the creation of the new field of medical bacteriology. His concepts. methods and discoveries are as fundamental to bacteriologists today as they were 100 years ago

Young Robert Koch Heullich Herrmann Robert Koch was born on December 11th 1843 In Clausthal, near Hannover The third child In a family which ultimately reached 13 children. he was the favourn€ child of his mother to whom he formed strong family bonds. In early childhood tle showed an Interest In natural science which was encouraged by his mother and his maternal uncie. Although an IndustriouS student with an aptitude for ma\t"lematICs and natural SCience, he was not outstanding wllh his o\iler studies. When It came 10 his univerSity career, Koch's Inclination towards natural science led him 10 sludy mediCine at Gottlngen. Here he was Influenced by Jaco~ Henle (1809 - 1885) who had written early and preslentiy about the germ theory of disease. Koch graduated with his doctorate In January 1866 at the age of 23 years and began a dl!f lcull phase of his life In general mediCine.

The young doctor Aller graduation Robert Koch decided to marry his childhood sweetheart Emmy Fraatz and thiS deciSIon meant he had to secure a source of Income to keep them both. For the next seven years Koc~' struggled to find a sUitable pract ice and adequate salary for his family, wh ich now Included his daughter Gertrud There were several moves, near penury and one year III the Prusslan Army In 1870. dUring the Franco-Prusslan war . However by 1873 he had settled In Wollsleln (Poland) and his private practice lees allowed him to create a rudimentary laboratory, Robert Koch's bnlliant research career In bacteriology started In tllese InauspicIOUS Circumstances al the 'advanced ' age 01 30 years (Figure 1).

Bacteriology hardly eXisted as a SCience at thiS time and the concept thaI microbes could cause disease was stili highly controverSIal. In spite of the works of LouIs Pasteur In the 1860's which had provided eVidence for the germ theory of disease.

Anthrax Koct,'s first researc~, prOject was to study anthrax because large and persistent outbreaks 01 the disease had occurred among farm animals In his locality, which Involved some human cases. Furthermore the large baCIllus, present In the animal blood, could be seen under his microscope thus making ItS study possible.

Figure 1: Robert Koch the young doctor.

HIs method 01 Investigation utilised microscope slide/cover-slip preparations, sealed with 011. which were mounted on a warm-stage first created by Koch. ThiS enabled him to study changes taking place on the slide, through the lens of his microscope. dUring Incubation. He used beef serum or aqueous humour from the eye-balls of slaughtered cattle, to cultivate the organisms. DUfing long, pallen! observation Koch discovered the highly resistant endospore formation by the organism which explained the persistence of the disease through Inteeleel soli .

USing InfectiOUS matenal from animals, he was able to cult ivate the organism through eight or more sub-cultures and then, on reinoculation Into animals, reproduce the disease. ThiS was the first proven work which used pure cultures and It demonstrated Koch's first prinCiple that specific organisms cause speCifiC diseases. Koch published thiS clasSiC work In December 1876 and immediately moved up Into the front rank of scientists In Germany,

The problems of reliably recognising the organisms under the microscope made Koch seek the help of Carl Weigert and Paul Ehrhch on stainIng solutIons which would help distinguish bacteria. He also collaborated With Ernst Abbe and Carl Zeiss to Improve both lens and sub-stage condensers. Out of this work came the first oil-ImmerSion lens which Improved microscopy enormously. USing the photographic expertise he had learnt from hiS uncle, Koch produced the first photomlcro· graphs of bactena.

Culture media Robert Koch's reputation lor research led to hiS Invltalton In 1880 to JOIn the Imperial Health Office In Berlin. ThiS move Into a research

Institute gave him aSSistants, co-workers and coJJeagues. Koch showed no problems In coping With thiS new environment: he soon acquired a small group of enthUSiastic researchers who would follow hiS lead,

He continued hiS studies on pure culture techniques and created hiS plating media uSIng eXisting broths to which he added gelalln_ Later on, agar was used In place of gelatin follOWing suggestions from his colleagues Fannie and Walter Hesse, Richard Petri changed the glass plate and bell jar technique of Koch to the 'Petri' diSh, which remains In use today. Fnedrich Loeff ler. another staff member, developed nutrient broth uSing peptone, meat extract and salt.

In creallng thiS technology of gelled, transparent media on which bar:lena could be spread With a sterile wire loop to form Isolated colonies of bacteria, Koch provided every laboratory with a Simple. consistently reliable method of obtaining pure cultures of bacteria. This tect,nique of bacterial Isolation has hardly changed In the 100 years Since It was developed.

KOCh 's postulates which created a diSCipline of proof for the relahonshlp of bacteria and their Infectious diseases, depended on reliable techniques of bacterial cloning. When Koch came to London In 1881 he demonstrated hiS plating method in Joseph lister's laboratories to Pasteur and a large audience. Pasteur congratulated him on his achievements but thiS was their first and best meeting, Subsequently relationships between these two great men soured with vltuperallve exchanges of communlcallOns. The Franco­Prusslan war undoubtedly played a role In creating the gulf between these two Intense patnots,

Tuberculosis Robert Koch began hiS work on tuberculosis In 1881 ThIS terrible scourge 01 Europe, the white plague, Infected onem-elghl of the entire population: one-In-three of the working population between 15 and 45 years of age USing hiS staining technique, Koch demon­strated tile tubercle bacillus In sputum and then grew the organism on coagulated serum prepared In glass tubes. The organism was then used to recreate the disease In animals.

Koch 's famous paper 'The Aetiology 01 TuberculOSIS' published In 1882 stands lor all time as a model of perfect SCientifiC procedure, He read hiS paper before a highly sceptical audience, led by Vi rchow the 'Lion of Pathol­ogy' who was convinced that consumption, phthiSIS and scrofulosis were totally different diseases. Step by step Koch destroyed the eXisting theones as he descnbed the progress of hiS work. The effect on the audience was stunning- there was no applause, no diS­CUSSion, no critiCism. Virchow left the meellng In Silence.

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r Cholera ThiS disease did not spread to the rest of the world until the 19th century. Europe suffered three major epidemiCS In that century; 1832 - 33, 1846 - 62 and 1864 - 75. When, In t 883, the disease broke oul again In Egypt both the German and French governments sent a team of Investigators to discover the cause of the disease and thus hopefully prevent 1\ spreading again Into Europe.

Robert Koch led the German team and soon discovered the cholera vibrio In the Intestines of Victims of the disease but tiWe more could be done as the epidemiC was waning and fresh cases were hard to find . Koch therefore took Ilis team to Calcutta where cholera was 51111 nfe. They quickly obtained pure cultures of the cholera organism and linked the disease to the common vehicle­dnnklng water. All animal inoculation tests were unsuccessful so that Koch's famous postulates remained unfilled but the constant aSSOCla\lon of the 'comma bacillus· In water and in the Inlestlnes of Infected patIents finally convinced the unbehevers. Koch returned In triumph to Berhn In 1884 to receive high honours from hiS Government.

Tuberculin-triumph and disaster Robert Koch continued his teaching and research In Berlin but his thoughts were dommated by a deSIre to find a cure for tuberculosIS. He secretly camed out work on extracts of tuberculOSIS cultures. which he Injected Into guinea pigs. The extract appeared to protect healthy pigs from Infection and It appeared to arrest the progress of the disease In Infected animals, Wlltl some of these animals recovenng completely. Koch was aware of the significance of his findings and Wished to continue his work Without publiCity. The German government. however, pressed him to publish his findings and In a paper entitled 'On Bactenologlcal Research' presented at the 10th International Medical Congress In 1890, Koch revealed that he had a substance which. In very small doses, would Inhibit the growth of tubercte bacilli In gUinea pigs.

ThiS stalement triggered a clamour for the substance. Berlin filled With anxious physicians and desperate patients. Koch refused to give Information about the reagent and could not supply enough materi al to satisfy the enormous demands. Rumours of corruption flew as the small volume of priceless reagent emerged from a laboratory controlled by Koch's son-In· law. There IS no evidence that Koch benefited by one pfennig from his discovery and his secrecy was caused by his fear itlat charlatan laboratories would not exactly copy his extraction procedure and thus expose patients to the nsk of Injections containing live. Virulent tubercle bacilli . The Heal th Ministry fully supported Koch throughout thiS difficult phase.

The tnals on human cases qUickly showed dubiOUS and dlsappolnllng resulls. TubercuJin proved to be a good diagnost ic reagent for tuberculoSIS but of httle value In treatment. Robert Koch left Berhn In 1891 With hiS reputat ion tarnished and took an eXlended vacation. On hiS return tle pubtlshed a paper fully describing tuberculin and liS method of manufacture

Whilst Koch·s publ iC hfe was under assault , a scandal In hiS prrvate life became the subject 01 common gosSIp. At 47 years 01 age Koch had become Infatuated With a young 17 year

Figure 2: Emmy Koch and Hedwig Freiberg.

finally left Berhn In 1893, follOWing her divorce from Robert and his subsequent marnage to HedWig In the same year (Figure 2).

The Robert Koch Institute In spite of hiS problems, the German government continued to hold Robert Koch In high esteem and In 1900 the Robert Koch Institute for InfectiOUS Diseases opened In Berhn (Figure 3). The Institute became a centre of excellence for clinical pathology and has remaIned so throughout the past 90 years.

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After Koch 's death, a wing of the bUildIng was made Into a museum and mausoleum to hold hiS body (Figures 4 and 5),

The closing years Robert Koch retired offiCially In 1904 but continued to travel widely overseas to study tropical diseases In man and animals. In 1905 tle received the Nobel Pnze In MediCine to complete the honours bestowed on him.

In 1908 he was asked to attend the inter­national meeting on tuberculosis held in

Figure 3: Robert Koch Institu te) or Infectious Diseases, Berlin .

old art student HedWig Freiberg. Emmy Koch Figure 4: Robert Koch Museum inside the tnstitute.

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Figure 5: Robert Koch Mausoleum.

Figure 6: Professor Robert Koc h near ret irement.

Discoverer Disease/organism Date

Koch Anthrax 1881

Koch Tuberculosis 1882

Pfeifler Bac!. Influenzae 1882

Loeffler Glanders 1882

Koch Cholera 1884

Loeffler Diphtheria 1884

Eberth Typhoid 1884

Rosenbach Stath Strep 1884

Escherich Bact. Coli 1885

Figure 7: Twenty gloriou s years of discovery.

Bibliog raphy Bac/enOiogy Science Tcc t1 nlcal Publishers. Madison, WI . USA

Brock T 0 (1 988) Rober/Koch A Lilem Mecflclncand Bulloct1 W (1938) TfleHlstoly ofBacteooiogy Oxford

New Culture Media Packs

Washington DC . There, Koch's Intransigent view about bovine tuberculosis caused him to become Isolated from the other representa· tlves. At the close of the meeting a member said 'Dr Koch Isolated the tubercle bacillus: today we have Isolated Dr Koch '.

Robert Koch returned to Germany a tired man and his heal th began to fail (Figure 6). He died at Baden·Baden on the 27th May 1910 at the age of 67 years.

Panegyric Space does not allow a full description of the many contributions that this brilliant. patient and meticulous worker made to bacteriology. Robert Koch 's greatest monument IS that he created order out of chaos. In the space of 20 years he. and others uSing his techniques, revealed the bacterial causes of all the major InfectiOus diseases which troubled mankind (Figure 7), an achievement which has never been equaHed since his time.

Discoverer Disease/organism Date

Neisser Gonococcu s 1885

Fraenkel Pneumococcus 1886

Bruce Malta Fever 1887

Weichselbaum Meningococcus 1887

Kitasato Tetan us 1889

Israel Actinomycosis 1891

Yersin Plague 1894

Ermengem Botulism 1896

Shiga Dysentry 1898

UP. ReprirlIed by Dover Publications Inc. New York Lechevaller. H A , and Solotorovsky, M ( 1965) Three CentufI(JS of Microbiology McGraw·HIII Book Company. New York

QXOId Ltd have Introduced SERVICE·PACKS. a new presentation of dehydrated cultu re media. SERVICE· PACKS consist of accurately p rewelghed quantities of powder presented In single use easy to open containers.

The contents of each container are added to the appropriate quantity of water and ster ilized as stated . No weighing is reqUired.

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A range of Oxoid cu ltu re media IS available In SERVICE­PACKS, in units sufficient to prepare volumes 01 media from 2 to 8 litres.

Each pack con tains 10 units.

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~8@&uD\0T3D POSITIVE •••

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BLOOD CULTURE SYSTEM