Microcalorimetric investigations on Listeria

Zbl. Bakt. Hyg. A 268, 15-23 (1988)

Microcalorimetric Investigations on Listeria FRANZ J. ALLERBERGER, ALISON SCHULZ, and MANFRED P. DIERICH Institut fur Hygiene der Universitat Innsbruck, A-6010 Innsbruck, Austria

With 6 Figures ' Received April 21, 1987 . Accepted May 20, 1987

Summary Sixty-one Listeria strains - Listeria monocytogenes (35 strains), Listeria ivanovii (8), Listeria innocua (7), Listeria toelshimeri (6) and Listeria seeligeri (5) - were tested microcalorimetrically for their heat production upon growth in Columbia broth. Listeria iuanouii and Listeria see/igeri displayed different and quite characteristic thermograms. Listeria u/elshimeri, Listeria innocua and all but one of the Listeria monocytogenes strains showed similar microcalorimetric curves, which differed considerably from those of Listeria ivanovii and Listeria see/igeri. Zusammenfassung 61 Listeria-Stamme - Listeria rnonocytogenes (35 Stamme), Listeria iuanovii (8), Listeria innocua (7), Listeria toelsbimeri (6) und Listeria seeligeri(5)- wurden mikrokalorimetrisch auf ihre Warmeproduktion bei Wachstum in Columbia-Broth untersucht. Listeria ivanovii und Listeria seeligeri ergaben charakteristische, jeweils eigenstandige Thermogramme. Listeria uielshimeri, Listeria innocua und alle Listeria monocytogenes-Stamme - ausgenommen Stamm SLCC1694 - zeigten ein gleichartiges, von den vorgenannten Arten differentes mikrokalorimetrisches Verhalten. Introduction The classification of Listeria has not been finished. Classification procedures used are biochemical fermentation characteristics, pathogenicity, hemolysis, serology and phage typing. Nowada ys the degree of relationship between Listeria is mostly ascertained on the basis of their biochemical behaviour and molecular biology (11). As a result of the development of highly sensitive calorimeters it is possible to measure the heat produced by bacteria during their multiplication and it is known that certain bacteria display characteristic thermograms (2). We wanted to find out if microcalorimetry would allow us to subdivide the Listeria and in case if such a subdivision would conform to one of the present classifications.

16

F.]. Allerberger, A.Schulz, and M. P. Dierich Materials and Methods

Microcalorimeter. We used two LKB calorimeters 2107/210 (Flow System). The heat producedby the growth of the bacteria is transformed into thermal currents bysemiconductors situated in measuring cells each with a capacity of 0.7 m!' Our machines wereadjusted to measure over a range of 0-30 ftV.

Table 1. Strains originating from the Special Listeria Culture Collection (= SLCq , Wiirzburg, F.R. Germany Strain (SLCC)

Serovar

L. monocytogenes

2371 696 811 1694 5048 5591 2715 5025 5581 151 reduced virulence 1191 reduced virulence 1688 reduced virulence 5779 rough 53 non-hemolytic

1/2a 3a 3b 3b 3b 3b 1/2a 1/2a 1/2 3a 3a 3b 1I2a 1I2a

L. ivanovii

2379 4121 4706 5336 5486 5755 5756 6737

5 5 5 5 5 5 5 5

L. innocua

3379 5224 6738 6743 6752 6765 6766

6a 3a 6b 6b 6b 6b 6b

L. seeligeri

3954 3503 3510 3858

1/2b 1/2b 1I2b 1I2b

L. ioelshimeri

5332 5333 5334 5828 5677 6100

6a 6a 6a 6a 1/2b 6a

Microcalorimetric Investigations on Listeria

17

Bacterial strains. The bacteria listed in Table 1 originated from the Special Listeria Culture Collection (= SLCq (Seeliger) and those in Table 2 were kindly donated by the State Bacteriological and Serological Laboratory Innsbruck (Semenitz). All strains were typed in tlfe Listeria Reference Laboratory Wiirzburg (Seeliger). Experimental method. In order to demonstrate the microcalorimetric pattern produced by the growing bacteria 5 ml of bacterial suspension (grown up from a single colony incubated for sixteenhours in 5 ml broth) were inoculatedinto 500 ml of broth, yielding an initial density of 106 bacteria/ml, The broth was then stirred continuously with a magnetic stirrer while incubatingin a waterbath (35Qq. After passingthrough the microcalorimeter at a rate of 25 ml/h the broth was collected In a separate container.

Table 2. Strains donated by the State Bacteriological and Serological Laboratory lnnsbruck, Innsbruck, Austria 1. monocytogenes

Strain

Serovar

originating from food LE39 austrian cheese LBO french cheese LE41 french cheese LE48 french cheese LE83 french cheese LE87 french cheese LE98 french cheese

428/86 427/86 146/86 147/86 149/86 237/86 238/86

l/2a 3b lI2a lI2a l/2a 1/2a lI2a

originating from adults suffering on - Meningitis K.M.42a female L.]. 73a male - Sepsis +E.R. 63a female

26/86 538/86 545/86

4b 4b 1/2a

originating from newborn infants P. male L. male B. male K. male H. female +0. male +S. male N. male P. male H. male M. male

70/86 74/86 99/86 108/86 268/86 268/86 272186 295/86 296/86 297/86 314/86

lI2a 112a lI2a lI2a lI2a lI2a lI2a 112a 112a 1/2a 112a

Strain

Serovar

148/86

6b

--

1. seeligeri -originating from food LE91 french cheese + = exitus letalis 2 Zbl. Bah. Hyg. A 268/1

18

F.]. Allerberger, A.Schulz, and M.P.Dierich Results

With the exception of Listeria ivanovii, Listeria seeligeri and strain SLCC 1694 all the Listeria examined- regardless of their antigenic pattern and capacityfor hemolysis - showed a double peaked thermogram with a common basic pattern (Fig. 1 and 2). This was clearly differentto the thermograms producedby corynebacteria, streptococci and staphylococci using the same procedure (Fig. 3). The thermograms of Listeria monocytogenes (except strain SLCC 1694), Listeria welshimeri and Listeria innocua showed the maximal thermal output 80 to 140 min after the first peak, and a varying transition back to the baseline at between 240 and 330 min after the first peak. The thermograms resulting from the eight strains of Listeria ivanovii, which were tested, had a uniform double-peaked curve, which differed considerably from all the other typesto Listeria tested (Fig. 4). Here the first peak showedthe maximalthermal output and the second peak followed 60 min later. The five strains of Listeria seeligeri which were tested also showed commoncharacteristic patterns in their thermograms (Fig. 5). They showed three peaks with a return to the baseline 360 to 460 min after the first peak, whichwas as high as or higher than either of the following peaks. Strain SLCC 1694, one of the 35 Listeria monocytogenes strains tested, displayed a microcalorimetric profile which was completely different from those showed by the 34 other Listeria monocytogenes strains (Fig. 6). The thermogramshoweda triple-peaked curve which returned to the baseline 300 min after reaching the maximal thermal output.

LL/'~ , ',-',

r,c: ~

..,.

I

,'0

110

i

i

'\

r~,,,,,, time

Fig. 1. Thermograms produced by: 1 = Listeria monocytogenes SLCC 53 (ahemolytic), 2 = Listeria monocytogenes SLCC 5779 (rough), 3 = Listeria monocytogenes SLCC 1688 (reduced virulence).

(hours)

Microcalorimetric Investigations on Listeria

1

1

/~,I\

c~/.

i

'------'---,1

"1~-----.------I

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0

I

,

rD~~~ ~

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1/", '" ' time

Fig. 1 == 2 == 3 ==

(hours)

2. Thermograms produced by: Listeria welshimeri SLCC 5332, Listeria innocua SLCC 6738, Listeria innocua SLCC 3379.

fl

1'"L .

~

'.

,1 0

i

\

2

'0

I-LJ.•• c·

Ii 3

~

\/ i i i

~

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,

10

time

Fig. 3. Thermograms produced by: 1 == Corynebacterium diphtheriae gravis NCTC 3984, 2 == Streptococcus faecalis ATCC 33186, ~ =

'\.t/1."J"",]nrnrr1U':

nrrro-u e

ATrr ? ~g? ~

(hours)

19

20

F.]. Allerberger, A. Schulz, and M. P. Dierich

l

-_/

/

//~\ I

s

I

~ I

i

10

r~, ,

q

JJJ. sec" 3

'0

time

(hours)

Fig. 4. Thermograms produced by: 1 = Listeria ivanovii SLCC 4706, 2 = Listeria ivanovii SLCC 4121, 3 = Listeria ivanovii SLCC 2379.

r,~ ,

to

r,~ ,

q

pJ.m·3~

I

.

. time

Fig. 5. Thermograms produced by: 1 = Listeria seeligeri SLCC 3510, 2 = Listeria seeligeri SLCC 3503, 3 = Listeria seeligeri SLCC 3954.

(hours)

Microcalorimetric Investigations on Listeria

21

/'~~

oJ ..,·

I / . ' " -r===

~ i

I

•

II

S

time

(hours)

Fig. 6. Thermogram produced by:

Listeria monocytogenes SLCC 1694.

Discussion By using a flow-microcalorimeter Delin, Monk and Wadso showed in 1969 that Escherichia coli produced characteristic thermograms and they suggested that it would be a useful technique in the characterisation of microorganisms (4). Boling, Blanchard and Russell reported on the use of micro calorimetry in the differentiation of bacteria in 1973 and two years later on the problems met thereby (2). The advantages of using Columbia broth for microcalorimetric investigations have been discussed previously (1).

Bacteria of the genus Listeria are fairly ubiquitous organisms. This group of grampositive, motile bacteria has long been regarded as one species, being subdivided into serotypes only according to varying surface structures. We examined the heat production of 61 different Listeria strains growing in Columbia broth. The measurement of the heat set free by the bacteria during multiplication allowed us to differentiate the Listeria strains we tested - with the exception of Listeria monocytogenes strain SLCC 1694 - into three microcalorimetrically different groups. Listeria ivanovii and Listeria seeligeri could be clearly distinguished both from one another and from all the other Listeria tested on the basis of their distinctive thermograms. Listeria monocytogenes (except for strain SLCC 1694 ), Listeria innocua and Listeria we/shimeri showed similar microcalorimetric patterns. In the currently accepted subdivision of the genus Listeria there is no strict correlation between the various serotypes and species except in the case of Listeria ivanovii and serotype 5 (12, 13). The thermograms of the eight strains of Listeria ivanovii, which we tested, were conspicuous by their uniform and characteristic pattern. For us this is especially noteworthy, since the taxonomic status of this group of Listeria is not regarded as firmly established (3). Listeria ivanovii was the only species of Listeria to show a correlation between its antigenic and microcalorimetric patterns. A rough strain of Listeria monocytogenes showed no irregularities either . Listeria seeligeri, established by Rocourt et al. as an independent species in 1983 , also differed from the other Listeria species in its thermogramal pattern; whereas structural analysis of the cell wall, analysis of somatic proteins, serology and phagetyping show no distinguishing characteristics which allow Listeria seeligeri to be classified as a separate species of Listeria (12). Listeria monocytogenes, Listeria welshimeri and Listeria innocua cannot be distinguished from one another on the basis of their thermogramal patterns. It is wellknown that the classification of strains into one of these three species on the grounds of their biochemical properties (capacity for hemolysis) is often problematic. Since Listeria

22

F.]. Allerberger, A. Schulz, and M. P. Dierich

innocua and Listeria welshimeri are generally regarded as apathogenic while Listeria monocytogenes is usually pathogenic, although among the organisms of the Listeria monocytogenes genus there is considerable variability in pathogenicity including complete loss of virulence, their species differentiation is of great interest (8, 9). Rocourt et al. have noted that there is only a 50% DNA homology between the species Listeria monocytogenes and Listeriainnocua, so there must be considerable genetic differences (11). However, these differences cannot be seen by the characteristics they display either on fermentation or on microcalorimetry. Which bacterial properties are responsible for the virulence of an organism remains unclear as yet. Nevertheless the production of hemolysin probably plays an important role (6). An ahemolytic strain of Listeriamonocytogenes (SLCC53) showed no special features on microcalorimetric, nor did the origin or pathogenicity of strains lead to any differences in the microcalorimetric profile (7). The Listeria monocytogenes strains with "reduced virulence" which we tested (SLCC 157, 1191, 1688) neither showed any differences in their thermal activity compared to virulent strains (9). The Listeriamonocytogenes strain SLCC 1694 was isolated from faeces in Denmark in 1963 (9). We could not find an explanation for its differing behaviour in microcalorimetry. The thermograms of all the Listeria strains tested microcalorimetrically differ quite clearly from those of staphylococci, streptococci and corynebacteria. In routine bacteriology there can set in difficulties in differentiating Listeria morphologically and serologically above all with enterococci and ~-haemolytic streptococci (3, 5). The taxonomy of Listeria is not yet finished; the microcalorimetric distinctiveness of Listeriaivanovii and Listeriaseeligeri could be an additional pointer to their classification as separate species.

Acknowledgment. We thank H. P. R. Seeliger and E. Semenitz for supplying strains and H. Hof for a critical reading of the manuscript.

References 1. Allerberger, F. J. und M. P. Dierich: Mikrokalorimetrische Untersuchungen an Salmonellen. Zbl. Bakt. Hyg. A 262 (1986) 462-473 2. Boling, E. A., G. C. Blanchard, and W. J. Russel: Bacterial identification by microcalorimetry. Nature 241 (1973) 472-473 3. Bortolussi, R., W. F. Schlech, and W. 1. Albritton: Listeria. In: Manual of clinical microbiology (E. H. Lennette, A. Balows, W. J. Hausler, and J. P. Truant, eds.), pp. 205-208. American Society for Microbiology, WashingtonlDC (1985) 4. Delin, S., P. Monk, and 1. Wadso: Flow microcalorimetry as an analytic tool in microbiology. Science Tools 16 (1969) 22 5. Dierich, M. P. und W, Kasper: Listeria-monocytogenes-Meningitis. Dtsch. Med. Wschr. 106 (1981) 837-839 6, Gaillard, J. 1., P. Berche, and P. Sansonetti: Transposon mutagenesisas a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 4 (1986) 50-55 7. Hof, H.: Virulence of different strains of Listeria monocytogenes serovar 1/2a. Med. Microbial. Immunol. 173 (1984) 207-218 8. Kluge, R. und H. Hof: Zur Virulenz von Listeria welshimeri. Zbl. Bakt. Hyg. A 262 (1986) 403-411

Microcalorimetric Investigations on Listeria

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9. Knorz, H. und H. Hof: Zur Pathogenitat von Listerien. Immun. Infekt. 2 (1986) 403-41 1 10. Recourt, f. and P. A. D. Grimont: Listeria welshimeri sp. nov. and Listeria seeligeri. Int. J. System. Bact. 33 (1983) 866-869 11. Rocourt, f., F. Grimont, P. A. D. Grimont, and H. P. R. Seeliger: DNA relatedness among serovars of Listeria monocytogenes sensu lato. Curr. Microbial. 7 (1982) 383-388 12. Rocourt, f. und A. Schrettenbrunner: Listeria seeligeri. Med. Welt 36 (1985) 1522-1 525 13. Seeliger, H. P. R. and K. Hahne: Serotyping of Listeria monocytogenes and related species. In: Methods in Microbiology (T. Bergan and ]. R. Norris, eds.), pp. 31-49. Academic Press, New York (1973) Dr. med. Franz Josef Allerberger, Institut fur Hygiene der Universitat Innsbruck, Fritz Pregl-Str. 3, A-6010 Innsbruck, Austria