* ERO-9- 72

US ARMY RESEARCH and DEVELOPMENT GROUP (EUROPE)Box 15 FPO New York 09510

MICROBIOLOGICAL RESEARCH IN.5PAIN:NOTES ON FIVE LABORATORIES

by

J..B. BATEMAN'

June 1972

JUL -26 ) 17il

EUROPEAN RESEARCH OFFICERoom 604, KeyIsign Hoou

429 Oxford StrietLondon W.I. ENGLANDRe~produced byNATIONAL TECHNICALINFORMATION SERV:CE

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-II

TECHNICAL REPORT

MICROBIOLOGICAL RESEARCH IN SPAIN:NOTES ON FIVE LABORATORIES

by

J.B. BATEMAN

CONTENTS

P ag e

I. INTRODUCTION ......... .. .................... 2

II. THE CENTRO DE INVESTIGACIONES BIOLOGICAS,MADRID ......... ... ...................... 2

a. Introduction ..................... 2

b. Instituto "Jaime Ferran" ...... .......... 3

c. Instituto de Biologfa Celular ..... ......... 7

III. UNIVERSIDAD DE BARCELONA .................... 9

a. Microbiology in the Faculty of Medicine .... 9

b. Microbiology in the Faculty of Science . . . . 11

IV. THE UNIVERSIDAD AUTONOMA DE BARCELONA,DEPARTMENT OF M'ICROBIOLOGY ... ............ 18

V. SUMMARY AND COMMENT ...... .............. 19

VI. REFERENCES ........ ............... 20

VII. DISTRIBUTION ....... ................... 23

VIII.ANNEX: The Seven Research Institutes of theCentro de Investigaciones Biologicas,Madrid ....... ................. . 25

I. INTRODUCTION

The choice of laboratories of microbiology to visit inSpain was made in several ways. The "Memoria of the Centrode Investigaciones Biologicas (1, 2) are a valuable guide tothe research interests of the center, and several of the sevenmember institutes touch on microbiology in one form or another.Correspondence had already been pursued in connection with onescientist who had submitted a research proposal. There was alsothe useful ONR (London) report of Dr. Hottle (3) and, finally,the on-the-spot recommendations of other scientists both inMadrid and Barcelona. No suggestion was made in these con-versations that any particularly important work in microbiologyis being done elsewhere in Spain although, for reasons of pro-fessional loyalty perhaps, the possibility was not actuallydenied.

It is not intended that this report shall duplicate thatof Dr. Hottle. I did talk with some of the same people,but the questions I asked led sometimes to discussion ofdifferent topics from those mentioned in his paper and wemay have received somewhilt different impressions.

II. THE CENTRO DE INVESTIGACIONES BIOLOGICAS,MADRID

a. Introduction

In the "Memoria" (1, 2) it is stated that under the lawof the Senior Council for Scientific Investigations, the Ramo'n yCajal Foundation is assigned responsibility for investigationsin medical sciences and animal biology, and for establishmentof an appropriate research center. Accordingly, seven insti-tutes were founded in 1969, with a 1970 strength of 237 tech-nical personnel, and a budget of nearly 89 million pesetasor about $14 million, mostly from the Ministry of Educationand Science. They occupy a large brick building designed tofit quite neatly into the acute angle formed by the inter-section of Velazquez and Joaquln Costa Streets not far fromthe center of the city. The satisfactory external shape isnot reflected internally by any features that would suggestintelligent planning for research laboratories. Space avail-able to an investigator is measured by the number of windows:dirty windows, it may be added, because the architect, seizedwith enthusiasm for doable glazing after a visit to Sweden,but faced with a frugal budget, modified the design so as tomake cleaning a major operation. In a building with perhaps300 windows, at a rough estimate, it takes two men an en-tire day to clean two of them.

2

The seven institutes, with their several departments andsections, are listed in the Annex. A little more inforr-tationabout the Institute of Genetics =d Anthropology and a dis-cussion of the work of the Section on Human Genetics will befound in another report (4).

b. Instituto "Jaime Ferr~n"

This Institute, headed by Professor Lorenzo Vilas L6pez,is really an institute of microbiology, with departments ofVirology, Bacteriology, General Microbiology and Protozoology;Professor Vilas acts also as head of General Microbiology.The various sections are listed in the Annex.

After describing the organization of the Institute andhearing something about the functions of the European ResearchOffice, Professor Vilas introduced the persons whose interestsmight, in his judgement, coincide with those of ERO: thesewere Dr. Miguel Rubio Huertos, head of the Department ofVirology and its section on plant viruses, Dr. Angel ProcopioGarcia Gancedo, head of the animal virus section, andMr. Antonio Portol6s Alonso, head of the section on thebiology of bacterial infections, Department of Bacteriology.This resulted in a discussion of a few particular subjectsrather than a run-down of all the activities of the Institute,which will be found listed in the "Memoria" (1, 2).

Immunodepressive effects of Pseudomonas aeruginosametabolites: Dr. Portolds has been interested in the anti-bioticresisistance of Pseudomonas and of clinical infectionswith the organism since about 1960. In 1963 he publishedthe results of a competently executed but rather pedestrianstudy of the inhibitory effect of 15 antibiotics singly, intheir 101 binary mixtures (certain mixtures of tetracyclineshaving been omitted), and in selected trivalent combinations,on 80 strains of P. aeruiinosa isolated from clinical infections (5).Following casual observation and similar reports in the literature,this led to examination of a possible relationship between theeffects of antibiotics and the nature and quantity of the pig-ments produced (6), using the techniques of Sephadex filtrationand elution of the extracellular liquids, automatic fractioncollection, and infrared spectroscopy on KBr pellets. Bythese means the various chromogens could be distinguished--pyocyanins, oxyphenoxazones and phenazines, in previouslyknown and in several unknown forms. These varied greatlyin nature and proportions with the concentrations andnature of the different antibiotics.

3

The foregoing investigations evidently provided a goodtechnical background for the current work on the biologicaland clinical significance of the soluble extracellular sub-stances produced by Pseudomonas, an immunosuppressiveeffect having been suspected by others because of the lowlevels of specific antibodies often found in human Pseudomonasinfections. Accordingly, extracellular polysaccharide,capsular substance and fractionated pigments were preparedand examined for their effects on the immune responses ofrabbits and mice to formalinized vaccines prepared fromEscherichia coli and Staphylococcus aureus (7). The testsmade included (a) measurements of bacterial agglutininresponse to challenge with the vaccines, (b) examinationof respiratory inhibition of bacteria by the immune sera,(c) measurement of the phagocytic activity of peritonealcells from immunized mice when incubated in vitro with the

extracellular Pseudomonas substances and 7U) the lymphocyticresponse and phagocytic activity of peritoneal macrophagesfrom mice injected with the test substances and subsequentlychallenged by intraperitoneal injection of live S. aureus.

The humoral responses appeared both by (a) theagglutination and by (b) the respirometric tests to bestrongly inhibited by the metabolites tested, but therewere discrepancies: the depression of respiratory in-hibition was seen to be about the same for all these sub-stances, while the suppression of agglutination was a gooddeal more marked with the polysaccharides than with the pig-ments. The cellular responses likewise seemed to be stronglymodified by the Pseudomonas metabolites. The production ofperitoneal cells in response to infection with S. aureuswas inhibited in both normal and vaccinated animals, aswas their phagocytic index. In the in vitro tests thephagocytic activity of cells from vaccinated animals wasinhibired by contact with the metabolites to values belowthose found in normal unvaccinated controls.

Thus the evidence for immunosuppresssive effects of bothpolysaccharide and pigments at humoral and cellular levelsseems quite good; further, the preparations were quite toxic(mouse LD 50s: polysaccharide 7.5, pienazines 25, phenoxazone12.5 mg/kg), though whether as a direct consequence of animmunosuppressive or other mechanism is not clear. In anycase I cannot grasp the need for the author's conclusionthat there are

4

"two types of immunosuppressive action:...an inert material inhibiting thenormal phagocytic activity (poly-saccharides) or as a cytotoxic sub-stance acting against the white cellsby interfering with their normalmetabolism (chromogens)." (7)

These conclusions do not seem to follow from the datapresented.

It is reported in the "Memoria" (ref 2, p. 60) thatan account of the Pseudomonas projects directed by Dr. Portol~s'section on the Biology of Infectious Bacteria has receivedtwo national awards, amounting to 60,000 pesetas ($858) fromthe firms Jorba and Pfizer. Dr. Portoles is continuing thiswork in an effort to identify the subcellular targets forcell wall endotoxin attack. He is now using the lysozyme--antilysozyme system for convenience as a replacement for thebacterial vaccines and is hoping to prepare 3 2P and 56Fe-labelled endotoxin. A preliminary study of the purificationof the protein-lipopolysaccharide complex has appeared fromDr. Rubio's department (8).

Research oiu African swine fever: Work on this importantvirus disease, which in Spain is comparable in its potentialeffects to foot-and-mouth disease in Britain, is being doneunder the direction of Dr. Gancedo in Dr. Rubio's departmentand with the latter's cooperation in electron microscopy.The major approach is in the search for an interfering virusor an infective non-pathogen, with the support of the necessarytechniques for cultivation and maintenance of this hexagonalDNA virus, for assay by cell culture and hemadsorption tech-niques, and differentiation from related virus diseases ofswine. My attention was directed to two papers, one dealingwith virus identification and the other with the search forinterfering viruses.

The first paper (9) introduces the use of pig kidneycells or established cell lines instead of the leucocytecultures used by other investigators. These cells wheninfected with African swine fever virus (ASFV) adsorberythrocytes, but the value of this in identifying ASFVis decreased by the fact that there is also some non-specific (control) hemadsorption. Some modest progresswas made in establishing ways of minimizing this non-specific component.

3 [

The second paper (10) examines the effect of infectionwith ASFV upon subsequent infection with a series of viruses:herpes simplex, vaccinia, eastern and western equine encephalitis,Newcastle disease, Sendai virus and porcine enterovirus PQlSl.The criterion used was the occurrence, or not, of cytopatho-logical effects of the challenging virus, and the infectedcells were primary porcine kidney cell cultures less thantwo weeks of age. Only in the case of herpes simplex virus(HSV) was any significant interference produced by priorinfection with ASFV. This interaction was confirmed inthree ways. First, the HSV in the cell suspensions previouslyinfected with ASFV was titrated in chick chorioallantoicmembrane (after an unstated interval, presumably, for growthof the HSV). The number of lesions was less by a factor offour than in uninfected controls. Second, the chick embryowas used instead of the porcine kidney cells and was infectedwith ASFV via the vitelline sac in eight-day embryos or theallantoic sac in ten-day embryos, followed at 12 days ofage by the HSV and two days later by excision of the chorio-allantoic membrane and counting of lesions. The interferenceproduced by ASFV was more pronounced when infection occurredat the earlier stage (eight-day embryo). Third, the needfor a sufficient interval between the two infections wasshown in more detail by infecting with HSV 3,4,5,6 and 7 daysafter ASFV. The HSV titer in the chorioallantoic membranedecreased smoothly by a factor of 3. This looks significant,but the results were reported graphically without any datafor the control group.

The results lead Dr. Garc'a to the conclusion that theAfrican swine fever virus can be included in the sametaxonomic group as herpes simplex. The program continueswith preparations for the titration of interferon in orderto determine whether the observed interference can be whollyor partially independent of the induction of interferon.A brief statement in the 1969 "Memoria" (ref 1, p. 67)refers to the finding that ASFV can induce productionof interferon active against both HSV and Sendai virus.

The work of the animal virus section also includes amore extensive study of the herpes virus and an attemptto find mutants. Antiviral substances are also beingexamined with herpes, vaccinia, and Sindbis viruses asthe targets (1, page 67).

Non-reverting L forms of pathogenic bacteria: Dr. Rubioreferred to the work of Dr. 1. Santaolalla Cerezo whosespecial interest has been in the lipopolysaccharides of

6

I

bacterial cell walls and in the L forms. Dr. Rubio statedthat by a long process of selection Santaolalla had beenable to obtain L forms of Bacillus tetani and Agrobacteriumtumefaciens which revert neither in culture nor duringinfection and that the infections produce the diseasecharacteristic of the parent microorganism. These L formsalso support the uptake and replication of bacteriophages.

The work on B. tetani seems not to have been published;Dr. Santaolalla has, however, spoken at several meetings,

including the X International Congress of Microbiology inMexico City, on the ultrastructure of L forms of A. tumefaciens,their relationship to the mycoplasmas, and their ability toproduce plant tumors without reversion. Dr. Ramona Beltra yMartinez de Vclasco has folloed this with a demonstrationof tumor production by the aseptic isolated nucleic acidsof phytopathogenic bacteria both in bean stems and incarrot-disc cultures (11). Since the tumors thus producedseem to satisfy accepted criteria of "authenticity" aslaid down by A. Braun in 1959, Dr. Beltra seems to havedisproved the contention that living cells of A. tumefaciensare essential for their formation.

Ultrastructure of viruses and virus-infected plants:Dr. Rubio, who worked in the University of California atBerkeley a few years ago, is primarily interested in thenature and ultrastructure of infected plants. Two papersmentioned here are easily accessible; they draw attentionto the presence of quasi-crystalline inclusions in plantsinfected with turnip yellow mosaic (12) and Petunia ring-spot (13) viruses, following upon Robley Williams' dis-covery in 1953 of crystalline inclusions of tobacco mosaic 1virus and the extraction of these inclusions from infectedcells by Rubio himself. Of special interest is theoccurrence of tubular crystals of circular cross sectionprobably formed by one mode of packing of the icosahedralvirus particles.

c. Instituto de Biologa Celular

This Institute, headed by Dr. David Vazquez Martlnez,has the Departments of Biochemistry (also headed byDr. Vazquez), of Cytology and of Cell Chemistry housedin the main building in Madrid, and, in addition, theDepartment of Microbiology at the University of Salamancaand the Department of Morphology and Physiology at theUniversity of Sevilla. The numerous research themesare listed in the "Memoria" (1,2) and can be classifiedroughly as follows:

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Biochemistry Mode of action of antibioticsRibosomesCell membranes: isolation andreconstitution

Cytology Fine structure of plant cellsI and cytology of cell division

Human cytogenetics

Cell Chemistry Glycolysis and gluconeogenesisin microorganisms and theirgenetic control

-Microbial degradation of hydro-carbons

Microbiology Mycology: lytic enzymes, proto-plasts, invertase excretion,and cell structure of fungi

Morphology andPhysiology Fifteen projects including

I enzymology of Chlorella,.chromosome replication inE. coli Hfr, carotenoidsynthesis in Phycomyces.

Mode of action of antibiotics: Without prejudging therelative merits of the various projects and the progress made,the Department of Biochemistry :seems to be the most activeand productive under Dr. ,Vazquez, perhaps because the effortis less diffuse than those of the others.

The discussion with Dr. Vazquez was mainly concernedwith matters arising from a preliminary contract proposalsub'mitted by him to the European Research Office some timeago and evaluated informally by Dr. Fred E. Hahn of WalterReed Army Institute of Research. Dr. Hahn recognized themerits of the proposal while offering certain suggestionsby which optimum scientific feasibility might be reconciledwith some degree of accommpdation towards the Army's in-terest in antibiotic resistant pathogens. A testimony tothe ifalue and reputation of Vazquez' work can be seen inthe plan of one of Dr. Hahn's colleagues to spend a yearin that laboratory. Unfortunately, aside from other factors,lack of space prevented this from being realized. I sawfor myself the overcrowded conditions in the laboratorydespite a slight recent expansion and conditions are notlikely to become tolerable foi a visiting scientist until

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the laboratory moves to a new building now under construction.This may not take place until 1974, when Dr. Severo Ochoa isexpected to move permanently from New York to Madrid.

The technical content of the Vazquez proposal will notbe discussed here since it is subject to modification. Itis sufficient to say that it deals with that class of anti-biotics that inhibit protein synthesis, and particularlywith the enzyme, peptidyl transferase, that catalysespeptide bond formation by transfer of a peptidyl groupfrom transfer-RNA to the aminoacyl-transfer-RNA of thegrowing polypeptide chain attached to the 70S ribosomecharacteristic of procaryotic organisms. The methodsused in localization of antibiotic action within theprotein synthesizing system have been reviewed by Vazquezet al (14) while Vazquez (15) has written a useful chapteron this and other modes of action of antimicrobial anti-biotics.

III. UNIVERSIDAD DE BARCELONA

a. Microbiology in the Faculty of Medicine

The Department of Microbiology is headed by ProfessorAgustin Pumarola Busquets who, while coping with routineresponsibilities in the areas of epidemiology, publichealth, microbiology and parasitology, and with a heavyteaching schedule, is able to run very effectively a researchlaboratory in which several epidemiological projects are beingcarried out. These have to do with leptospirosis, respiratoryviruses, arboviruses and enteroviruses. Without immediateiccess to his publications, 1 can only report briefly myconversation with Professor Pumarola.

Leptospirosis (Weil's Disease): The disease is ofconcern in Spain both in human beings and in pigs. Infectionsin man have been associated with migratory workers in therice fields who start the season in the south and work theirway northwards as the harvest date advances, Since the firstextensive epidemiological study in 1947, the followingmeasures have been taken, or attempted, to control theinfection:

1. Vaccination: The narrow spectrum of only threeserotypes in Spain (in contrast to Italy where there areabout 10) has made possible preparation of a goodrjrecipitated vaccine. It is particularly useful andeffective because it can be administered exactly onemonth in advance of the harvest date when infection

9

will occur. All workers who have been in the fields formore than five years have some degree of acquired immunity.

2. Chemotherapy: Penicillin is very effective ifgiven for the first six to 10 days of the harvest. Inother countries tetracyclines have given better results.Administration of penicillin produces shock, of diagnosticvalue, by release of toxin from the spirochaetes. Shockis severe but transitory, with complete recovery in 24 hours.

3. Eradication of rats: This has met with littlesuccess.

4. Treatment of fields with calcium cyanamide toproduce a low pH unfavorable to the survival of Leptospir a .A transitory benefit.

5. Mechanical harvesting.

6. Drainage: Replacement of rice paddies wherepossible by orchards. This is being pursued in the provincesof Vnlencia, Alicante and other parts of Andalusia, but willhave some impact on the economy because of the importanceof rice as a crop for exportation. The results have beengood and leptospirosis is disappearing, for example, fromthe Ebro delta in Tarragona.

The infection of pigs with Leptospira pomona introducedfrom France is of economic importance, causing-abortion in80% of the cases.

Arthropod-borne virus infections: Evidence for Tahnyavirus infections is said to Rave been found in Spain follow-ing the epizootic occurrence in the Rh6ne delta. No otherarboviruses are known; ticks are plentiful but no tick-bornedisease has been identified. Boutonneuse fever occurredbefore the Civil War but is now unknown.

Respiratory virus infections: Pumarola maintains theWHO influenza reference center. Results of a survey intropical areas have been sent to the Communicable biseaseCenter in Atlanta. Similar surveys of influenza and re-lated respiratory viruses are now being done in childrenand in out-patients.

Enteric viruses: Isolates of a number of entericviruses from infants were obtained and sent Zi year ago toAtlanta for examination, but without any response to date.

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b. Microbiology in the Faculty of Science

The Department of Microbiology is housea under theroof of the large, labyrinthine, inelegant but spaciousbuilding of the Faculty of Science within easy walkingdistance of the Ramblas. I was told that I could notsee the head, Professor Ramdn Pares Farras until aninconveniently late hour despite a 5 o'clock appointment,but a colleague, Dr. J. Sancho, was kind enough to explainthe program, with frequent disclaimers of any authorityto interpret Professor Pares' views.

The general impression one had was that of anenthusiastic and capable group of professors and studentsengaged in challenging problems but handicapped by lackof modern equipment.

According to Dr. Sancho, the two principal lines ofinterest are in microbial taxonomy and genetics.

Microbial taxonoay: Numerical taxonomy is beingcombined with biochemical taxonomy and serological typing.A small IBM 1130 computer is available in the laboratory;they also have access to the faculty IBM 360 machine,although this is too small, and the possibility of usingthe City computer housed in the Town Hall. Experimentalwork is being done, including the recording of thermaltransition ("melting") curves of DNA, using a painstakingmanual procedure for lack of money.

Microbial genetics: Episomal information transfer:The main line of work during the past ten years, publishedin a series of papers in the Catalan and Spanish languages,has been the study of an organism with a particularly labileform of DNA from the point of view of episomal transfer ofcharacteristics to other organisms. An illustration wasgiven in Hottle's paper (3) in the induction of the abilityto secrete glutamic acid in strains which normally do notdo so. According to Sancho, Pares considers this to bean example of a general phenomenon which will be foundto account, in part, for the complexities encounteredin the classification of microorganisms. He asserted,rather cautiously, that the mechanism is not necessarilyconfined to closely related organisms; typical entero-bacteriae have been transformed into typical members ofthe genus Achromobacter and more ambitious inter-familytransfers to the pseudomonads are being attempted. Sanchosaid that Pars is preparing an extended version, in English,of his investigations and his views, for presentation at

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the furthcoming National Congress of Microbiologists in Madrid.The work has been followed closely by Dr. J.L. Ingraham of theUniversity of California at Davis, where Pares' co-worker,J. Guinea, is now working.

It may be of interest, because of the lack of Englishpublications, to sketch the background to these recentfindings as given in an incomplete set of reprints fromPares' laboratory (16-23) based upon doctoral theses byS. Hern~ndez, R. Clotet, and J. Guinea. Reference willnot be made to the papers individually as a lot of ,.ross-checking was necessary in order to arrive at a consistentpicture of what was done, and how.

Production of amino acids durn bacterial growth inliquid medium: It seems that the work began with aninterest in the production of amino acids during bacterialgrowth, already studied especially by Kinoshita in 1959,who drew attention to the possibility of amino acidsynthesis on an industrial scale and to the questionsthat must be asked about the disturbance of biosyntheticregulatory mechanisms that must underly amino acidaccumulation. Pargs and co-workers first screened anumber of isolates, which they carefully defined taxo-nomically, by growing them on a medium containing onlyglucose, ammonium chloride, and salts. They measuredchanges in dry mass, soluble protein and amino acidsduring growth over periods up to 264 hours: solubleprotein by turbidimetry of the trichloracetic acid pre-cipitate, amino acids by spectrophotometry of the coppercomplex, corrected for the ammonium chloride in the medium.During the lag phase, all cultures showed an initial appear-ance of amino acids and soluble protein caused by lysis(autolysis) of a substantial fraction of the inoculum.Thereafter, throughout the logarithmic phase (and beyond,according to the graphs presented), ia all isolates butone, the amounts of amino acid and soluble protein perunit dry mass remained constant: amino acids were not"secreted" but were present in the medium solely as aresult of the slight constant rate of autolysis. Thisbehavior was typical of E. coli irregular; a Flavobacterium(3 strains), a member of the Achromobacter-Alcaligenes group,and Bacillus megaterium. The single exception was a strainC3 of Citrobacter intermedium (or Escherichia freundii,E. intermedium) described as'being taxonomically intermediatebetween Escherichia and Aerobacter (Klebsiella?). This hadthe unique property of releasing amino or many hoursbeyond the lag period, reaching final concentrations of

12

&,A

Fi

about 3.2 mM/liter* or a free amino acid nitrogen to proteinnitrogen ratio of about 67. The absolute rate of accumulationin the medium was dependent upon several factors; optimumvalues were found for the ammonium chloride concentration ,(aided by the presence of solid calcium carbonate) and forthe size of the inoculum. Nitrogen balance calculationsshowed that under optimum conditions 74% of the nitrogenpresent as amino acids, proteins, and cell sediment is amino:acid, although the data presented also show that a substantialpart of the inorganic nitrogen lost from the medium was notrecovered, casting some doubt on the numbers but not on thequalitative fact. The inoculum size had a very pronouncedeffect- within a certain ranje,.the tate of. amino acidproduciion was independent or size at inoculum, whileover a lower range it increased to a maximum value. Thisbehavior led to the speculation, certainly on rather scantyevidence, that two modesof amino acid synthesis were in-volved. Some support was derived from paper chromatographywhich showed that at the low (inoculum-independent) ratethe product was 44% alanine, increasing to 71% at tfhe highrate. The remainder in both cases was mostly glutanic acid,with some aspartic and some basic amino acids.

Identification of amino acid secreting clones by bio-autographic assay: More detailed information about thediffering rates of amino acid production was obtained bystreaking the cultures on agar containing.the standardmedium and using bioautographic procedures for detectingamino acid production by the individual colonies'. A specificdetector for glutamic acid is the glutamic acid requiringstrain of Leuconostoc mesenteroides P-60 (ATCC 8042)a suspension of which in agar containing "glutamic. assay,broth" is poured over the colonies of C. intermedium andincubated. For detection of alanine, a Suspension ofPediococcus cervisiae (ATCC 8081) can be used in the samemanner using "bactoalanine assay medium," with 'somewhatless satisfactory results.

When a suspension of C. intermedium is plated 'andassayed for glutamic acid in this manner, about 40-50%of the colonies become surrounded by a cloudy zone ofL. mesenteroides, showing the presence of glutamic acid

*This figure may be compared to the maximum of 224.0 reportedby Samejina (cited in ref 17) for an unidentified gram-negative bazterium secreting L-alanine.

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("A colbnies"), while most of the renainder are clear*("C colonies") with a few ("B colonies") that show smallzones of;comparatively faint cloudiness. Since the B coloniesare in the minoTity, it is argued that the phenomenon is notsimply due to statistical, variati'on which would lead to a.Poisson distribution with maximum numbers of B colonies,but a real~differentiation into secreting and non-secretingcells. Generally similar results were reported with thealanine detector, the alanine positive c6loniet being alsoglutamic acid positive, as shown by distributing parts ofsingle, colonies on fresh plates. However, alanine wasnever absent and the distinction between A and C wasnever as clear cut as with glutamate.

These results might suggest a preliminary scheme:

A B C colony types

S ? factorss +1 S- iS- colony types

S being used to 4enote an hypothetical factor responsiblefor amino acid secretion and s+,, st and s- a more informativeway of describing the colony types.

'At tmpts' to modify co'lony type by adding differentsubstrates or intermediary metabolites: , The importantobservation was made', but not reported in detail in thepapers cited here, that by additioh of .- ketoglutarate'to the medium all the cells 'could be converted into aminoacid producers so that all colonies were of the s+ type.,On the other hand, the proportion of -+ and s- colonieswas unaltered by addition of glyoxalate, glutamic acid oralanine, or by replacing the glucose in the medium' by apentose, glyrerol or citrate. These observations weretaken t6 imply that the cells that give rise to s- coloniescontain an active repressor of isocitrate dehydrogenaseWhile s+ colonies arise from cells 'in which the repressoris inactivated:

*It is stated that even the clear' colonies, examined inthe microscope, show the occurrence of micro-colonies ofthe detector organism, so that glutamic acid is nevertotally absent..

14

NADP NADPH

CIT - ISOCIT .- KET----GLU

-CO2

isocitratedehydrogenase

T R

Some support for this scheme was found in measurementsof isocitrate dehydrogenase in cells harvested from agarslants that had been inoculated with cell suspensionscontaining different proportions of s+ and s- colonyformers (obtained by the acridine orange method described

below). The isocitrate dehydrogenase activity was an 'approximately exponential function of the percentage +of s colony formers, being about twice as great in s

cells as in s-. One might perhaps have expected a greaterdifference in view of the large differences in glutamateproduction- -perhaps about seven-fold--so that importantquestions remain unanswered. Par6s assumes that thereplication of the inactive represssor is controlled byan episome integrated in the bacterial chromosome, probablynear the locus of a regulator gene. Thus the postulatedfactor S may be identified tentatively with this episomein its integrated state, S(int).

The anomalous ("paradoxal") effect of acridineorange: The episomal hypothesis of amino acid secretionwould imply that (a) some cells would contain the autonomous(cytoplasmic) episome S(aut), (b) the episome would beabsent from some cells, and (c) by analogy with otherwell established cases, S(aut) could undergo both intra-cellular transformation to S(int) and intercellulartransfer by conjugation. Some of the evidence for theexistence of S(aut) was obtained by experiments withacridine orange (AO), which is known to inhibit thesynthesis of extrachromosomal DNA.

The anomalous effects of AO consist in the contrastingresults of growing small inocula for short times* and

*AO lO10g/ml, inoculum 1 - 100 cells/ml, incubation 12 hours.

15

large inocula for long times.* The first, if repeated,eliminates all s+ colonies; the second eliminates the s"colonies; the second eliminates the s" colonies.** Ifeither of these are subsequently grown without AO, thedistribution of colony types reverts to the "normal"40-50% s . Finally, availability of cultures withdifferent proportions of s and s' cells permitscalculation of generation times, given as 0.9 hoursand 1.25 hours respectively.

Pares uses the ideas outlined in the precedingsection to explain these findings. Piecing thingstogether, I think the argument runs somewhat as followsthough rather differently expressed: Since the pro-portions of s+ and s" colonies are amenable tomodification, the two forms of the episome S must existin some sort of "equilibrium" or steady state:

S(auto) S(int) I ,

In addition, transfer of the episome can occur from s"cells containing S(auto) to s cells lacking S:

Cell 1 + (Cell 2)(nS)- (Cell 1)(n t S) + (Cell 2)(n"S) (2) .

In very dilute cultures containing AO, we are not con-cerned with reaction (2) because it depends upon cell-cell encounters which will occur rarely. In this situation,since AO inhibits replication of S(auto), the pool of S(auto)will be gradually attenuated, favoring "disintegration" ofS(int) by reaction (1), so that the final result of continuedgrowth under AO should be the disappearanc of s+ coloniesand the elimination of the episome. The s colonies doindeed disappear. Par~s does not predict the eliminationof the episome, nor does he discuss in this connection itsreappearance when the cells are restored to a medium freeof AO. He does, however, state without evidence that thenumber of S(auto) particles per cell is very large. It

*AO l0/.g/ml, inoculum 104 106 cells/ml, incubation 12-72 hours.

**Table 1 ref 18 showing the results of the first harvest fromAO medium substantiates the decrease of s at low inocula butdoes not show the claimed increase at high inocula unlesstwo AO experiments that were run without controls are included.

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is i

is qualitatively conceivable therefore tha the s+ coloniesare completely eliminated before the episome disappears,so that under the conditions of these experiments "recovery"of s+ status can occur. The experimental test of this wouldbe to increase the number of transfers under AO beyond the100% s" condition.

With large ipocula and long incubation periods,the increase of s colonies is taken to prove theexistence of factor S :.n the integrated state and toshow that in this condition it is not sensitive to AO.This, alone, does not explain the phenomenon, and Pare'shas recourse to the calculation showing that s cellshave a selective advantage because of their shorter .4generation time, supplemented by a supposed effect of AOin increasing the velocity (probability?) of reaction (1)1in favor of S(int). Nothing is said about reaction (2)under these circumstances but it can be imagined thatthis occurs with increased frequency in dense suspension5prior to the attenuation of S(auto) thus increasing thenumber of cells containing S(auto) and thus potentiallyable to be transformed to the s+ state. Careful calculationof the quantitative consequences of these mechanisms areevidently needed, and possibly a critical re-examinationof the experimental basis.

Episomal conjugation sensitive to mechanical agitation:So far the phenomena described do not depend qualitativelyupon reaction (2) although this, if it occurs, must influencethe distribution of S and therefore the colony types. Directproof of intercellular transfer by conjugation lies in thesensitivity of the colony type distribution to mechanicalagitation, referred to several times but not described indetail in the papers available to me.

Interspecific transfer of ePisome: In principlereaction (2), intercellular transfer of free episome,could occur bctwcun species. This would be an efficientmeans of incorporating new material in the genome ofanother cell because of the supposed rapid intracellularreplication of S(auto) and the _igh probability ofintegration, estimated at 3xlO per cell x generation.

Success was claimed in transferring the amino acidsecretion factor S(auto) from C. intermedium C3 toParacolobactrum intermedium (ATCC 1166), which hadBeen tested for amino acid production with consistentlynegative results. Mixtures of the two organisms witha ten-fold predominance of P. intermedium were incubated

17

for two hours at 300C, diluted, And streaked .n McConkeyplates. Since P. intermedium ferments lactose only veryslowly, the colnies were white while those of C. intermediumwere pink and thus easily distinguishable. When theP. intermedium colonies were resuspended, streaked onagar, incubated for 25-30 hours, and overlaid with Leuconostoc,colonies positive for glutamic acid were found in five experi-ments out of six, involving 47 s+ colonies out of a total of464. Thus interspecific transfer seems to have been demon-strated. Some of the secreting colonies were transferred,grown for 24 hours, and again plated for amino acid assay.Of 200 colonies so examined all were non-secretors, ,s.This rapid elimination of the S(int) episome casts somedoubt upon the interpretation, if not the accuracy, ofthe original observations that seemed to show interspecifictransfer of the ability to secrete glutamic acid.

Conclusion: This brings us back to the starting point,where we reported the rather far.-reaching views expressedon the possibilities of information transfer amongcomparatively remotely related microorganisms. Thebrief survey just completed, without reference to thework of other laboratories, has left me with the feelingthat there are many loopholes which make the episomalinterpretation of the data somewhat less than completelysatisfactory. In the hope that this may be due to theincompleteness with which the data have been abstractedfrom the unavailable doctoral theses, the news that Paresis preparing an authoritative summary is very welcome.

IV. THE UNIVERSIDAD AUTONOMA DE BARCELONADEPTMENT OF MICR 0oBIO.OGY

The Universidad Aut6noma is one of three such newuniversities in Spain, the others being in Madrid andBilbao. Recent decisions by the Ministry of Educationare intended to place all the Spanish universities, thenew and the old, on an equal footing, with some libertyand autonomy in a narrow sense. The new university inBarcelona is in its fourth year. It is housed in a con-verted hospital building, an uninspired but spaciousstructure in the grounds of the Santa Cruz Hospital atSan Pablo. In a pleasant new temporary building nearbythe Department of Microbiology, now three years old,occupies the first (upstairs) floor. It is headed byProfessor A. Foz with two associate professors and sixresearch associates with doctorates in pharmacology.biochemistry and microbiology. Professor Foz was kindenough to describe his research interests which centeraround the subject of brucellosis.

18

V}

Brucellosis is prevalent 3pain, with at least 6000human cases. Professor Foz, 20 years experience inthis field, is a WHO expert in Lne diagnosis and serologyof Brucella, including antigen standardization and inter-pretation of the Coombs test for B. abortus. His U.S.connections include Dr. Wesley Spink of the Universityof Minnesota Medical School, who has visited the Department.

Problems of diagnosis arise in patients who havereceived vaccination against cholera (over a million inSpain), because their sera contain antibrucella agglutinims.Professor Foz is investigating the nature of these anti-bodies in relation to those occurring in brucellosis.Another microorganism, Yersinia enterocolitica, relatedto Pasteurella pestis, also causes formation of anti-bodies active against Brucella. The triangular set ofVibrio-Brucella-Yersinia relationships is being studied,including some work on the non-pathogenic Vibrio Naq whichis found to be antigenically quite different fromV. cholerae. Other rganisms causing urinary and entericinfections are being studied; the salmonelloses nre verycommon, and shigellosis rather uncommon, in Spain. Allthis is being done in close collaboration with theMunicipal Hospital for Infectious Diseases.

Antibiotic testing: A new antibiotic, tobramicina,is being tested for the Lillie Company. Foz finds allstrains of Pseudomonas to be very sensitive in vitro.The first in vivo tests are being made in theU.S.

This appears to be an active young department whoseaccomplishments have perhaps received less than theirproper recognition in the outside world because of thelanguage difficulty; Professor Foz, a friendly andcourteous man, speaks French well but is uncomfortablewith English.

V. SUNARY AND COMMENT

A few of the microbiological research projectsengaging the attention of scientists in Spain arereviewed, in varying depth, on the basis of visitsto five laboratories and perusal of some of theirpublications. Topics discussed include: (1) Theimmunosuppressive effects of metabolites of Pseudomonas(Instituto "Jaime Ferrgn", Madrid). (2) Searh forviruses which interfere with infection by Africanswine fever viruses (same). (3) Pathogenic non-reverting L-forms of pathogenic bacteria (same).

19

(4) Locali:,ati.on of antibiotic action within the protein

synthesizing system (Instituto de Biologia Celular,Madrid). (5) Control of leptospirosis (Universityof Barcelona, Faculty of Medicine). (6) Interspecifictransfer of episomes for amino acid "secretion"(University of Barcelona, Faculty of Science).(7) The Brucella-Vibrio-Yersinia cross-reactingsystem ("Autonomous" Univerity of Barcelona).

The projects appear to be carried out with muchcompetence although it is likely that they would benefitby closer and more frequent contact with ongoing researchin some of the major centers, none of which can be saidto be situated in Spain. In addition to a certainisolation, the microbiologists work under crowded con-ditions with inadequate support for modern equipmentand technical help. On the other hand, social mobilityis less marked than elsewhere and a well-trained assistantis likely to remain available as long as the positioncan be maintained financially.

The microbiologists visited were informed aboutthe functions of the European Research Office andseveral expressed interest in submitting informalresearch contract projects for further discussion.Negotiation of research contracts on some of thetopics listed above could be beneficial to the U.S. Armyresearch program while assisting the Spanish scientificcommunity in a modest way, both materially and inpromoting increased contact with scientific establish-ments in the United States and elsewhere. Commentsand expressions of interest from the recipients ofthis report are cordially invited.

VI. REFERENCES

1. Patronato Santiago Ramo"n y Cajal: Centro deInvestigaciones Biologicas. Memoria 1969. (Madrid).

2. ib., Memoria 1970.

3. Hottle, G.A. 1971. Microbiology in Spain. Officeof Naval Research, London, Report R-11-71, 30 March.

4. Bateman, J.B. 1972. Surveys of Thalas.emia andOther Blood Defects in Spain, European ResearchOffice Technical Report 7.

20

5. Portols, A., and Perez-Ure~'a, M.T., 1963. Controlof infections by Pseudomonas. Proc. III Internat.Congr. ChemOther., C-47, 9-522. Georg ThiemeVerlag, Stuttgart.

6. Portolds, A., Espinosa, M., and Hidalgo, A. 1971Penicillin and polymyxin effects on the chromogenesisof Pseudomonas aeruginosa strains. J. Antibiotics,

266-269.

7. Perez Urena, M.T., Espinos, M., L6pez, R., andPortol6s, A. 1972. Immunosuppressive effectsproduced by some extracellular metabolites fromPs. aeruginosa strains. Zbl. Bakt., in press.

8. Rubio, N., and Lopez, R. 1971. Purification ofPseudomonas aeruginosa endotoxin by gel filtrationon Sepharose 4B. JChromatog., 57, 148-150.

9. Gil-Fernandez, C., Alonso, L., Garcla-Gancedo, A.,

and Ronda, E. 1971. Hemadsorcion inspecifica yespecifica sobre celulas de ri~on de cerdo normalese infectadas con el virus de la peste porcinaafricana. Microbiol. Espa? ., 24, 23-38.

10. Gancedo, A.G., Alonso, N.L., Gil-Fernandez, G.,Ronda, E., Vilas, P., and Ronda, C. Undated proof.Interferencia inducida por el virus de la pesteporcina africana (PPA). "Memo 112," pages 1-6.

11. Beltra, R. and Rodriguez de Lecea, J. 1971.Aseptic induction of the crown-gall tumors bythe nucleic acid fraction from Airobacteriumtumefaciens. Phytopath. Zeitscir., 70, 357358.

12. Rubio-Huertos, M., Vela, A., and Lopez-Abella, D.1967. Crystalline arrays of spherical particlesin turnip yellow mosaic virus-infected cells.Virol., 32, 438-444.

13. 'Rubio-Huertos, M. 1968. Further studies onultrastructure of plants infected with Petuniaringspot virus. Protoplasma, 65, 465-476,

14. Vazquez, D., Staehelin, T., Celma, M.L., Battaner, E.,Ferna*ndez-uTfoz, R., and Monro, R.E. 1970. Peptidebond formation in procein biosynthesis and theaction of antibiotics. FEBS Symp., 21, 109-130.

21

15. Vazquez,-D. 1969; Mechanism of action of antibiotics.Ann. Rep. medicinal Chem., Chap. 15; 156-169.(Acadiemic Press, New Yo-r-.).

16. Clotet. i Ball6s, I ., and Par's i Farr'a1s, R. 1967.Dos tipus d6 segregaci6 d'aminoacids per uncoliform(Escherichid intermedia (3). TreballsSoc. Catalana Biol., 11 1-9

17. Cl6tet, R., Guinea, J:., and Pares-Farr'as, R. 1968.Segregacion de aminoacidospor una cepa deC. intermedium. Microbiol. 'espa5., -21, 15S-173.

18., Guerrero, R., 'and Pares i Farr~s, R. 1969.Effecte paradoxal del taronja d'acridina enrelacji6 a 1'accic% d'un factor episo'mic deCitrobacter intervedium C3.. Tz'eballs Soc.Cgtalana R~io. 7P 873-87.

'19. Urge11, J.B., and Pare's i Farr~s, R. 1969.Activitat Isocitricdeshidr ogen~sica en preparatssolubles de dif ferents poblacions, de Citrobacterintermedium C3. ibid. , 91-97.

20. Clotet, R., Guinea, J., and Par6-Farrafs, R. 1969.Produccio~n'de alanina por fermentacion conCitrobacter intermedium C3. Revista espa~i.

21. P ar '-Farr~s, R., and Guinea, J. 1969. Transferenciainterspecifica del factor episomico que condicionala segregacion de glutamatlo en Citrobacter intermediumC3. Anales Estacion exp. Aula DLI,77-2-ff T

22. Guinea, J. 1970. Analisis colonial de la produccionde acidoiglut-amico por Cit'robacter intermedium C3.Mic~robil. espai. , 23, 1322

23. Eerna"ndez,'S. 1970. Heterogeneidad c,)1onial deCitrobacterintemedium C3, en relacion con lasegregracionde ha . ibib., 23, 139-147.

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VII. DISTRIBUTION

1 ERO, CO2 AFEB (Prof G.J.Dammin; Col B.W. Prior)1 AMEMB London (Dr. A.G. Mencher)1 AMEMB Paris (Dr. E.L. Piret)1 AMEMB Rome (Dr. O.H. Ganley)9 AMRDC (Col. R.F. Barquist (2)); Brig Gen R.R.Taylor (2);

Maj R.M. Glickman; LTC D.W. Sample; LTC J.L.Hatfield; LTC G.B. Randolph, Jr; Col J.B. Young)

1 BAMvC (LTC B.A. Pruitt,, Jr)2 BSRL (Dr. J.E. Uhianer; Dr. J. Zeidner)1 CRL6 DARD (Gen C.D. Daniels, Jr, ARE-A (2); ZA; Dr. C.

Lamanna ARL-M; Col G. Rapmund, ARL; Dr. E.M.Sporn, ARL-P

3 EOAR (LTC R.F. Prueher; LTC R.B. Wallace;Maj W.H. Inge, Jr)

k FASEB (Dr. C.J. Carr; Dr. E.L. Hess)1 FDA (Dr. R.A. Littleford)1 MEDEDCG (Brig Gen R.M. Hardaway III)2 MRL (Director; Dr. C.C. Hassett)1 NAMRU-3 (GO)1 NASA2 NATO (Dr. E.G. Kovach)I2 NBL (Dr. R. Valeri)2 NBRL (Dr. G.A. Hottle)2 NEPMU-7 (Cmdr W.G. Miner)I NIH (Dr. M.D. Leavitt, Jr)A3 NSF (Dr. E.R. Sohns (2); Dr. B. Bartocha)5 ONRL (Dr. E.I. Salkovitz; Dr. R.R. Sonnenschein;

Dr. J. Foss; Capt A.J. van Tuyl, Jr; Dr.A.W.Frisch)2 OTSG (LTG H..B. Jennings, Jr) .1 RDRD1 RDRF (Col B.C.A. Walton)1 RrRL (Mr. H.F. Weiler)2 RIEM (Col L.G. Jones)2 USAARL (Col R.W. Bailey; Dr. E.J. Baldes)1 IJSAMLO (LTC M.C. Duffy)1 USAMvIRNL5 tJSANL (Dr. S.D.. Bailey; Dr. H.L. Jacobs;

Dr. G.R. Mandels; Dr. J.F. Oesterling;Dr. D.H. Sieling)

5 ISARIID (Col W.R. Beisel (3); Dr. A. Buzzell;2 USPHS Col D. Crozier)2 USPHS(Dr. R. Freckleton; Dr. B. Blood)

1 WHO (Dr. W.P. Faulk)

23

19 WRAMC-WRAIR (LTC C.R. Angel; Dr. M. Artenstein;Dr. J.F. Barbaro; ClM.E. Conrad;LTC R.T. Cutting; Dr. B.L. Elisberg;Dr. E.H. Fife, Jr; Dr. A.D. Glinos;Dr. F.E. Hahn; LTC H.C. Holloway;Col T.H. Lamson; Dr. H.E. Noyes;LTC R.A. Qisson; Col W.E, Rothe;Col P.K. Russell; Dr. E.H. Sadun;LTC P.K. Hildebrandt; Dr. T.R. Sweeney;Dr. S. Vivona)

1 AMRDC (Col K.R. Dirks)

24

ANNEX

The Seven Research Institutesof th-e

Centro de Investigaciones Biologicas, Madrid

(abridged and translated from ref 2)

Institut "Santiago Ramnv yCa.'al"

Department of Biophysics

Cytology and Comparative HistologyElectron MicroscopyComparative NeuroanatomyNeuroangiologyPathological Anatomy of the Nervous SystemHistochemical Laboratory

Instituto "Jaime Ferran"

Department of Virology

Plant VirusesAnimal Viruses

Department of Bacteriology

Biology of Bacterial InfectionsSymbiotic Nitrogen FixationDirect Nitrogen FixationBacterial. Physiology and Genetics

Department of General Microbiology

YeastsMicrobiological TechniquePhytopathological MicrobiologyMicrobial ChemistryApplied Mycology

Department of Protozoology

Protozoal CytologyEcology

25

instituto "Arnaldo de Vilanova"

History of Spanish Medicine

Instituto "Gregorio Mara-6n"

Department of Metabolism

Pancreatic HormonesImmunochemistry of HormonesMolecular BiologyMolecular Genetics

Department of Experimental Endocrinology

ThyroidSteroidsBiology of Photosynthetic Organisms

Instituto de Biologia Celular

Department of Biochemistry

Biosynthesis of ProteinsBiochemistry of Membranes

Department of Cytology

Cell StructureCell Physiology

Department of Cell Chemistry

Microbial PhysiologyEnzymatic Regulation

Instituto de Enzimologfa

Department of Enzymatic Regulation

BiocatalysisMetabolic Regulation

Department of Microbial EnzX!g

Biochemical GeneticsBiosynthesis of Enzymes

26

A

Instituto de Geneytca y Antropologia

Department of Anthropology

Human SomatologyHuman Genetics

Department of Dynamic Genetics

Population GeneticsDevelopmental Genetics

Department of Cytogenetics

Plant CytogeneticsAnimal Cytogenetics

2

I

27 I>

-. -I J'