Zbl. Bakt. Hyg., 1.Abt, Orig. A 251, 505-511 (1982)

1 Unite d'Ecologie bacterienne (Prof. H. H. Mollaret) , lnstitut Pasteur, Paris, France'Institut fiir Hygiene und Mikrobiologie (Prof. H.P.R.Seeliger), Universitat Wiirzburg,FRG

Isolation ofBacteriophages from Listeria monocytogenes

Serovar 5 and Listeria innocua

Isolierung von Bacteriophagen aus Listeria monocytogenes Serovar 5und Listeria innocua

JOCELYNE ROCOURTt, ANGELIKA SCHRETTENBRUNNER 2, and HEINZP. R. SEELIGER 2

With 1 Figure' Received September 19, 1981

Summary

11 new bacteriophages, 3 from Listeria monocytogenes serovar 5 and 8 from Listeriainnocua were isolated from lysogenic strains without induction.

Phagovar determinations were carried out with these phages and 12 others isolated fromL. monocytogenes serovars 1/2 and 4 b. 76% of the 142 strains from different serovars testedgave a lytic pattern. The new phage set increased the percentage of determinable L. innocuastrains to 61.7% .

Different phage patterns underline the distinction between L. monocytogenes and L. in­nocua . L. monocytogenes serovar 5 is characterized by its great sensibility to many phagesfrom lysogenic strains of various serovars.

Zusammenfassung

11 neue Bakteriophagen, 3 von Listeria monocytogenes Serovar 5 und 8 von Listeriainnocua wurden ohne Induktion aus lysogenen Stammen isoliert. Mit diesen Phagen undmit 12 anderen aus Listeria monocytogenes Serovar 1/2 und 4b frtiher isolierten wurdenPhagovar-Bestimmungen durchgefuhrt,

76% der 142 untersuchten verschiedenen Listeria-Serovare zeigten ein lytisches Muster.Der neue Phagensatz errnoglichte, den Prozentsatz der bestimmbaren Listeria innocua­Starnme auf 61,7% zu steigern. Das unterschiedliche Muster der Phagen hebr den Unter­schied zwischen Listeria monocytogenes und Listeria innocua hervor. Listeria monocyto­genes Serovar 5 wird durch ihre grolie Sensibilitat gegeniiber vielen Phagen IysogenerStamrne verschiedener Serovare charakterisiert.

506 J.ROCQurt, A.Schrettenbrunner, and H. P.R.Seeliger

Introduction

Prev ious investigations on bacteriophages of Listeria monocytogenes (2, 3, 4, 6,7, 8, 9, 13, 17, 21, 22, 23) have primarily dealt with strains assigned to serovars1-4 in the classical schema of Paterson, extended to its pre sent form by Donker­Voet and Seeliger. In recent years two important serovars have been added to theabove schema : the strongly hemolyzing strains (now serovar 5), first detected byIvanov in Bulgaria from clinical cases of listeriosis in sheep (12) and the non­hemolytic listeriae (sero var 4 f, 4 g, 6 -, no w Listeria innocua) , the latter mainlyrecovered from fecal specimens and other environmental sources (19, 20). Theincreased isolation rat e of L. innocua is due to the improved isolation techniques(5, 16) fro m heavily contaminated specimens.

The above mentioned two biogroups of the Genus Listeria have not been in­cluded in previous studies on Listeria - phages. The phage-typing schema recentlyproposed by Audurier et al. (2,3) is not applicable to the se two biogroups, becausethe host specificity of each phage is closely related to the serovar of the lysogenicstrain (2, 7, 21) and these groups were not included. The following report there­for e extends the previous studies of Audurier et al. to L. innocua and L. mono­cytogenes serovar 5.

Material and Methods

1. Bacterial strains

All Listeria strains employed in this study belong to the Special Listeria Culture Collec­tion of the Institute of Hygiene and Microbiology of the University of Wiirzburg, GFR.They are therefore characterized according to standardized methods (18). The strains wereisolated from different specimens (Table 1) in 14 different countries (further details areavailable on request).

2. Phage assay test

Bacteriophages were isolated from lysogenic strains of L. monocyto genes serovar 5 andL. innoc ua without induction according to previously described methods (2) : The filtrate(Millipore O.4S JI) of a 6 h culture in tryptose phosphate broth (Difco) was applied to thesurface of a tryptose agar plate (Difeo). The plates were previously flooded with thepropagating Listeria strains in the log phase of their growth . Incubation followed at30 °C overnighr. Each phage was purified 3 times by repeated single plaque propagation.The phages were assigned by the number of the lysogenic strain from which they wereisolated. Propagation was carried out according to the agar layer method described byAdams (1).

All strains were tested with phages for their RTD (routine test dilution) and 100 x RTD.Lysovar determination was performed with the newly isolated phages and with otherspreviously found in L. monocyt ogenes (2, 3, 4): phages 1967, 2685, 4477, 575, 1652 and12029 from serovar 1/2 and the phages 2671, 1444, 2425, 3551, 3552, 1317 fro .n serovar 4b.The phage solutions were applied on tryptose agar plates with the Lidwell-apparatus (14).The reactions were read after an incubation of 18 h, at 30 °C.

Results

The application of 49 filtrates of L. monocytogenes serovar 5 stra ins and 47filtrates of L. innocua on 118 different strains of the genus Listeria yielded 11 new

Bacteriophages from Listeria monocytogenes 507

Table 1. Source of the 142 tested Listeria strains

Origin Samples Listeria monocytogenesserogroups1{2 4b 5

L. innocua

6a 6b

Human

Animal

Environment

No information

Total

C.S.F::·BloodPost-abortumFecesOthersNo information

C.S.F.BloodPost-abortumFecesOthersNo information

714

4156

1

3

2

43

2

11

304

10

4

52

16

322

1

23

47

"C.S.F.: cerebrospinal fluid.

phages, 3 from L. monocytogenes serovar 5, and 8 from L. innocua. The propa­gating strains were chosen according to the serovar of their lysogenic strains. Theresults of phagovar determination of 142 Listeria strains with the 11 new phagesand the 12 previously by Audurier et a1. isolated ones (2, 3) gave no correlationbetween the lysovar pattern observed and the source and geographical origin ofthe Listeria stains. The conventional phage set allowed to determine only 84.6010of serovar 5 strains and 10.6 % of L. innocua strains in this study, whereas the11 new phages lysed 92.3010 of serovar 5 strains and 61.7010 of L. innocua strains.The isolation of the new phages did not increase the percentage of susceptiblestrains of serovars 112 and 4 b (a single 4 b strain of the 23 tested was lysed byL. innocua phages, while ir was not attacked by phages of serovar 4 b!).

Fig. 1 demonstrates the relations between Listeria - phages and Listeria sero­vars: the phages of serovar 5 attacked 48 (92.3 0(0) of 52 strains of serovar 5 anda few (6 of 47) strains of L. innocua. It still has to be clarified whether the threephages isolated from serovar 5 are identical or not, as their lytic spectrum onstrains of serovar 5 and L. innocua is very similar. All the phages of L. innocualysed 47 (90.4 0(0) of 52 strains of serovar 5, as well as 15 (83.3 0(0) of 18 strainsof L. innocua 6b, three of the phages (4211,4286,5375) attacked 14 (48.2010) of29 strains of L. innocua 6 a. None of the phages isolated from serovar 5 and L.innocua gave a lytic pattern on serovar 1/2 strains. Serovar 1/2 was lysed only bythe phages isolated from its own serovar. It is noteworthy that a phage (1317)of serovar 4 b lysed L. innocua and two phages (4211,4286) isolated from serovar6 a and 6 b lysed strains of serovar 4 b. Serovar 5 was very sensible to many phagesfrom several other serovars. This pattern seemed to be specific for this group ofstrains.

508 J.Rocourt, A. Schrettenbrunner, and H. P. R.Seeliger

,,,IIIIIIIIII•

42114285537542764277

~4292

4295337

IIIIIIIII•

ILISTERIA MONOCYTOGENESI

1/2 a zbb -,----.,.-----c

t2350----------....J3885--------------14719 ---J

Fig. 1. Relations observed between Listeria phages and Listeria strains of various serogroups.------- lysogenic phages------ lytic activity of each phage

In conclusion, two groups of phages were found (Table 2):

- specific phages whose lytic spectrum was restricted to the serovar of the lyso­genic strains.i. e. for serovar 1/ 2: phages 1967,4477,575,1652, 12029

for serovar 4 b: phage s 1444,2425,3551,3552,2671- non specific phages which acted on different serovars

i. e. from serovar 1/ 2 : phage 2685from serovar 4 b: phage 1317and all the phages isolated from L. monocytogenes serovar 5 and L. innocua.

Discussion

The phages described in th is paper were isolated from spontaneously lysogenicstra ins. Induction is recommended for research purposes on Listeria phages (6, 7,13, 21) but was not attempted in this study in order to eliminate the productionof bacteriocins (10, 11, 15). Such bacteriocins may interfere with the reactions inthe lysovar determination . A high proportion of the strains belonging to L. innocuacould not be assigned to a lysovar, A similar situation was noted among strains

Bacteriophages from Listeria monocytogenes 509

Table 2. Specificity of Listeria monocytogenes and Listeria innocua phages

Phages Serovar of the lysed strainsSerovarof the a 6alysogenic strain n° 1j2b 4b 5 b

c

a 1967 +1j2b 4477 +

c 575 +1652 +

12029 +2685 + +

4b 1444 +2425 ..L

3551 "1

3552 +2671 +1317 + + +

5 2350 + +3885 + +4719 + +

6a 5375 + +b 4276 + +

4277 + +4292 + +4295 + +5337 + +4211 + + +4286 + + +

of serovar 1/2 (2, 3, 4). Therefore the use of spontaneously lysogenic strains maynot be appropriate for the isolation of certain Listeria phages.

Since the phage titres after the agar layer propagation method were found tobe sufficiently high (108- 1°) to allow lysovar determination with an RTD valueof 10-4, no further attempts were made to use adsorption cofactors (ions, aminoacids). The newly isolated phages did not increase the percentage of the lysovardeterminable strains among serovar 1/2 and 4 b, but they lysed 61.7 '010 of all L.innocua strains tested (instead of only 7.4 010 with the conventional phage set (3)).

The strains of L. monocytogenes serovar 5 were noted for their high percentage(92.3 010) of susceptibility to phages. They were furthermore lysed by phage 2685of serovar 1/2 and by phage 1317 of serovar 4 b and surprisingly by all eight phagesobtained from L. innocua.

The above findings appear to introduce a new dimension into the determinationOf Listeria lysovars: phage 2685 of serovar 1/2 attacks serovar 5; phage 1317 ofserovar 4 b lyses serovar 5 and L. innocua. The phages of serovar 5 attack L. inno­cua, while phages of L. innocua attack both serovars 4 band 5. These finding needfurther clarification by extended examination of more strains. The phages obtainedneed to be characterized more closely by appropriate methods.

33 Zbl. Bakr. Hyg., I. Abt. Orig. A 251

510 j .Rocourt, A.Sch retrenbrunner, and H.P.R.Seeliger

An attempt to correlate the phage sensitivities to the breakdown of sugars (API50 E System, La Balme-les-Grottes , F- 38390 Montalieu-Vercieu, France) wasunsuccessful. Likewise no correlation between the lysovar pattern observed andthe source of the isolate could be establi shed. This is in line with previous con­clusions of Sword and Pickett (21). These observations point to the possibilitythat together with other phenotypic characteristics, they could be indicators for aspecial taxonomic position of serovar 5 within the Genus Listeria.

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[ocelyne Rocourt, M. D., Unite d'Ecologie bacterienne, Institut Pasteur, 25 rue duDr Roux, F-75724 Paris cedex 15, France