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Typing of Listeria monocytogenes by monocin and phage receptors
International Food
Microbiology
Journal
of
31 (1996) 245-262
Typing of Listeria monocytogenes by monocin and
phage receptors Elizabeth Bannerman,
Patrick Boerlin, Jacques Bille*
Centre National de Rkfkence des Listhias, WHO Collaborating Center jbr Foodborne Listeriosis, CHUV, 44, rue du Bugnon, 1001 Lausanne, Switzerland Received
20 July 1995; revised
11 January
1996; accepted
7 February
1996
Abstract One hundred strains of Listeria monocytogenes from both sporadic and epidemic cases were typed by monocin production combined with phage receptor and reverse phage receptor methods. The monocin-phage combination gave 72 types with 100% typability and 97% reproducibility. The results were compared to those of serotyping, phage typing, ribotyping, multilocus enzyme electrophoresis, restriction enzyme analysis and RAPD (random amplification of polymorphic DNA). The monocin/phage types were comparable in terms of discrimination with other methods for epidemiological investigations. The index of discrimination of using the monocin typing and phage receptor/reverse phage receptor method combination (0.99) for both the 87 epidemiologically unrelated strains and the epidemiologically important serogroup 4 strains was the highest of the seven different methods analysed. This combination of methods was simple, highly discriminatory and reproducible and can be carried out in a non-specialized laboratory. However, like most of the other Listeria typing methods, both the method and the indicator test strains need to be standardized. Keywords:
Listeria
monocytogenes;
Monocins;
Phage receptors
1. Introduction Listeriocins * Corresponding
0168-1605/96/$15.00
(Tubylewicz, author:
1963a,b)
Tel: 41 21 3144057,
0 1996 Elsevier
PII SO168-1605(96)01003-3
or monocins
Science
(Hamon
fax: 41 21 3144060.
B.V. All rights
reserved
and P&on,
1962, 1963)
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E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
are bacteriocins of the genus Listeria and were first described by Sword and Pickett (1961). Monocins are resistant to trypsin, and are inactivated at 50°C and at pH 4.5. Monocins have been described as defective phages without heads (Bradley and Dewar, 1966) with antibiotic-type effects against Listeria and other Gram-positive organisms (Ortel, 1989). The monocins with this bactericidal effect are thus comparable to other bacteriocins such as colicins, pyocins, or staphylococcins. Bacteriocins of genera other than Listeria have found application in epidemiological studies as specific markers for bacteria (Tagg et al., 1976). Typing schemes have been established based on either the production of, or the sensitivity to a range of different bacteriocins. Hamon and P&on (1963) found 10 different types of monocins and Tubylewicz (1963b) classified 4 types, both after ultra-violet (UV) induction of Listeria strains. Ortel (1978) described 6 different monocins after mitomycin C induction of L. monocytogenes strains. Tagg et al. (1976) suggested that greater discrimination for typing may be obtained by the combination of bacteriocin production with bacteriocin sensitivity, or by either or both bacteriocin-based methods used with other typing methods, such as serotyping or phage typing. The combination of bacteriocin production and sensitivity was useful for typing streptococci (Kelstrup et al., 1970) but was found not to be discriminatory for subtyping Listeria (Curtis and Mitchell, 1992). The combination of bacteriocin typing with serotyping of Listeriu was also poorly discriminatory (Wesley and Ashton, 1991). Recently, Lebek et al. (1993) combined phage typing (phage receptor and reverse phage receptor analysis) with the monocinogeny and demonstrated a relatively high discriminatory power among some Listeria strains. Among the typing methods available for L. monocytogenes, serotyping (Seeliger and Hiihne, 1979) and phage typing (Rocourt et al., 1985) are relatively simple and cost-effective. Serotyping, however is not discriminatory enough for epidemiological studies, while phage typing, though discriminating, is limited because not all Listeriu strains are phage typable (Rocourt et al., 1985; Espaze et al., 1989). Multilocus enzyme electrophoresis (MEE) is discriminatory (Boerlin and Piffaretti, 1991) and also allows estimation of genomic relationships among strains (Piffaretti et al., 1989). This method however, is time-consuming and labour intensive. Restriction enzyme analysis (REA) is easy to perform and discriminatory (Nocera et al., 1990; Wesley and Ashton, 1991; Nsrrung and Gerner-Smidt, 1993) but the electrophoretic patterns obtained are often difficult to interpret and like the MEE needs electrophoresis equipment. Ribotyping has also been successfully used to type L. monocytogenes (Graves et al., 1991; Nocera et al., 1993) but this method is also labour intensive and not discriminatory enough, particularly for the clinically significant serovar 4b strains (Nocera et al., 1993). The most recent typing method is the random amplified polymorphic DNA (RAPD) analysis (Mazurier and Wernars, 1992) which seems to be highly discriminatory (Boerlin et al., 1995). Like the other molecular methods, it needs special equipment and reproducibility problems may sometimes be encountered (Boerlin et al., 1995). Another molecular
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
241
typing method currently used but not evaluated here, is pulsed-field gel electrophoresis (PFGE). Brosch et al. (1994) demonstrated that PFGE can also be used to divide Listeria into genomic groups which in turn can be used to correlate serotypes. Listeriocinogeny combined with phage receptor and reverse phage receptor analyses, though time consuming, can be carried out in any laboratory and does not need special equipment. In an attempt to determine whether in a case of an epidemic, this combination of techniques could replace any of the other more cumbersome methods in a non-specialized laboratory, we compared the approach of phage typing in combination with bacteriocin typing with 6 other Listeria typing methods for discriminatory power and for utility in epidemiological studies.
2. Materials and methods 2.1. Bacterial strains
The same set of strains used by Boerlin et al. (1995) and consisting of 100 isolates of serovars 1/2a (19) 1/2b (8) 1/2c (3) 3a (1) 3b (1) 3c (2) 4a (1) 4b (61) 4c (I), 4d (1) 4e (1) and 7 (1) were used in the present study. Except for phage typing which was done in Paris (Rocourt et al., 1985) all strains have been previously typed in our laboratory (Boerlin et al., 1995). With the exception of the 12 L. monocytogenes serotyping reference strains from the Special Listeria Culture Collection (SLCC; Wiirzburg, Germany), the strains selected were isolates from human (75) animal (3) food (8) and environmental (2) origin. Serovar 4b strains which are frequently encountered in human listeriosis and which are less distinguishable by other methods are particularly well represented in this study. Serovar 4b strains analyzed included 7 phage type I and 6 phage type II isolates from the Swiss epidemic of 1983-1987 (Bille, 1990). Also included were epidemiologically related mother/baby isolates and isolates from different sites of the same patient. All indicator strains (7 isolates for monocin testing and 21 isolates for phage testing; Lebek et al., 1993) were obtained from the Swiss Federal Office for Public Health. Berne. 2.2. Typing methods 2.2.1. Monocin analyses
Monocin typing was carried out using the technique described by Lebek et al. (1993) but with 5 instead of 14 spots per Petri dish (8.5 cm diameter). 2.2.2. Phage receptor analysis
The multiplication described by Lebek eleven indicator (lstrains for serogroup
of bacteriophages in Listeria was induced by mitomycin C, as et al. (1993). All the strains were subjected to this test using 11) strains for serogroups l/2 and 3 and ten (12-21) indicator 4. L. monocytogenes serovar 7 which does not belong to either
248
E. Bannerman et al. /ht.
J. Food Microbiology 31 (1996) 245-262
group, was tested with both sets of indicator strains. It was important that the flooding of the plates, as well as the subsequent decanting was unidirectional 2.2.3. Reverse phage receptor analysis The reverse phage receptor analysis was carried out on all 100 strains. Using the same technique described for the phage receptor analysis, the strains to be typed were taken as indicator strains and the lysogenic strains (1 to 8 for serogroups l/2 and 3; 12 to 20 for serogroup 4) served as the test strains (Lebek et al., 1993). 2.3. Reading of the tests All the plates were read against a dark background or with an oblique illuminating apparatus. For monocin tests, this enabled the zones of inhibition and for the phage tests, the presence of phage plaques to be clearly seen. For the phage tests, reactions were considered positive when plaques of phages (usually with tailing) were seen. Clear well-defined zones were considered negative. The results were always confirmed by a second reading after the plates were stored overnight at 4°C. 2.4. Reproducibility tests All 100 strains were typed at least twice. The tests were all repeated after 4-6 weeks. Strains with discrepancies were subjected to two additional analyses; the fourth test was for confirmation. For the two phage tests, all non typable strains, the epidemiologically related strains and L. monocytogenes serovar 7 were also tested three times with both sets of indicator strains. 2.5. Index of discrimination (DI)
The index of discrimination Hunter and Gaston (1988).
was calculated
using the formula
proposed
by
3. Results 3.1. Monocin typing
The identification of the different types of monocins (Table 1) was based on that of Lebek et al. (1993) for listeriocin testing. Three different zones of inhibition (large transparent, small transparent and large diffuse) were observed. As a result of their monocin reactions, all the L. monocytogenes non-serogroup 4 strains, that is, L. monocytogenes serogroups l/2, 3 and 7 will be referred to as group A strains and the serogroup 4 (serovars 4a, 4b, 4c, 4d, 4e) as group B strains. Four monocin types (I, VIII, IX, and XII) were found among the 35 isolates of the group A strains with 22 (62.8%) strains belonging to type IX. Seven strains of this group were not typeable with the 7 indicator strains used in the monocin test (Type I = NT).
I = NT VII X XIII I = NT VIII IX XII
Serogroup 4(65 strains)
Non-serogroup
NT, not typeabie. -, resistant (no zone of inhibition). 1, large and transparent. 2, large and diffuse. 3, small and transparent.
4(35 strains)
Monocin type
L. monocytogenes
Table 1 Identification of the monocin types
7 1 22 5
2 1 60 2
No. of strains
1 1 1
1 1
a
3
2 2 2
b
3 3 3
C
2 2
d
e
f
Susceptibility of indicator strains to different monocins g
2
F
250
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
However, all 7 strains were typeable by either the phage receptor or the reverse phage receptor methods. Reference strain, serovar 3b (SLCC 2540) was the only strain which showed reproducibility problems with the monocin test. Two additional tests, however, gave the same readings as the first test and the strain was eventually identified as a type VIII. Four different monocin-types (I VII, X and XIII) were found among the 65 isolates of the group B strains with 60 strains (92%) belonging to type X. Two reference strains, SLCC 2374 (serovar 4a) and SLCC 2376 (serovar 4c) did not produce monocins and were assigned to type I. All the results were reproducible for this group. The reproducibility for monocin typing was thus 99% and the overall typeability was 91%. 3.2. Phage typing The scheme used for interpreting the phage analysis tests is shown in Table 2a (serogroups other than 4) and Table 2b (serogroup 4). The criteria of Lebek et al. (1993) were used for reading of the plates. Of the group A strains, 18 (51.4%) and 22 (62.8%) out of 35 strains were typeable with the phage receptor and the reverse phage receptor tests, respectively. The 65 group B strains (Table 2b), gave 40 (61.5%) and 58 (89.2%) typeable strains, respectively, with the phage receptor and the reverse phage receptor tests with the set of indicator strains used. Reproducibility problems were encountered with the same two strains (BE 1050 and SLCC 2755) for both tests, leading to an overall reproducibility of 98% for both the phage receptor and the reverse phage receptor methods. Among the 35 group A strains, 15 different types were identified with the phage receptor test while 30 different types were found among the 65 group B strains. The reverse phage receptor analysis gave 17 and 26 different types for groups A and B, respectively. 3.3. Combined results When the three methods were combined, 72 different types were obtained (Table 3) and the overall typability was lOO%, with 97% reproducibility. The seven isolates belonging to the Swiss epidemic type I strains (sv 4b, ribovar I, ET 1, REA-A, lysovar 47/108/340/2389/2425/2671/3274 and RAPD type 2) were indistiguinshable by the three methods (type 36, Table 3), as well as by the six other methods. Likewise, the six Swiss epidemic type II isolates comprised a single type using the three methods (type 69, Table 3). However, the Swiss type I strains (type 36) were differentiated from the Swiss type II strains (type 69) by the combined monocin/phage methods. Two isolates from the same patient but obtained from different sites (SG 6415 = blood and SG 6416 = ascites; type 39, Table 3) could also be paired together using the present three methods in combination with the six other methods, previously described. The same could be done with a mother/ child pair of isolates (ZH 7415 and ZH 7416; type 8, Table 3). For the 87 epidemiologically unrelated strains, the number of types and the index of discrimination (DI) obtained with each method are shown in Table 4. The
+ + +
+ t
+ +
+
+
+ + + t
+ + t +
+
6 6 6 7 7 I 8
6 6a 6b 7 la 7b 8
+ t + t + + +
+
t
5
5
+ t + t + + +
+
+
+ + + + + + +
+
+
+
+
+
+
H
G
NT A Al A2 A3 B Bl 82 B3 84 C Cl D Dl E El F FI
4
11
4
10
+
9
+
8
3
7
3
6
2
5
2
4
0 1 1 1
3
NT I la lb
2
Code
strains
No. of plaques
Code
Indicator
4 phage types
Reverse
4 phage types
4 and (b) serogroup
Phage receptors
non-serogroup
(a) non-serogroup
for interpreting
for interpreting
(a) Diagram
Table 2 Diagram
8
I
6 6
5 5
3 3 4 4
2 2 2 2 2
0 1 I 1
No. of plaques
phage receptors
+
+
+
+
t
+ +
+
I -
t
+
+ +
+ + +
t + +
+
3
strains
+ + f
t + +
+
+
2
Indicator
+
4
+
t
+ + + +
+
+
+
+
+
5
6
+
+
+ + + + + +
+ + +
+
7
+
+
+ + + + +
+
+
+
8
for interpreting
2
3 3
4 4 4 4 5 5 5 5 6 6 6 6 6 1 7 I 8 8 8 8 9 9 9 10
13
14 14a
15 15a 15b 15c 16 16a 16b 16~. 17 17a 17b 17c 17d 18 18a 18b 19 19a 19b 19c 20 20a 20b 21
reaction.
0
NT 12 12a
+ , positive
No. of plaques
Code
Phage recrptors
(b) Diagram
Table 2
f
+ +
+ + +
+
+ + + +
+ +
+ +
12
+ + + + + + + t + +
+
+ +
13
Indicator
serogroup
+ + + + + + t +
+ + + + +
+
+ + +
+
14
strains
+ + + +
+ + +
+
+ + + +
+ + + + + + +
+
15
4 phage types
+ + + + +
+ + + + +
+ +
+ +
+ +
+ +
+
16
+ + + + + + + + + + t +
+
+
17
+ +
+ +
+ +
+ +
+
+
+
18
19
+ + + + + + +
+ + +
+ + +
+ +
i + +
+
20
-
+ + + + + + + + + + + + +
+ + + + + + + t + +
+ +
+
21
T
s
R RI
42
z1
NT L LI L2 M Ml M2 N Nl N2 0 01 02 03 P Pl P2 P3
Code
Reverse
9
0 1 1 1 2 2 2 3 3 3 4 4 4 4 5 5 5 5 6 6 6
No. of plaques
phage receptors
+
+
+ +
+ +
+ + +
+ +
+
12
i-
+
+ +
+ + + + + + +
+
+
13
Indicator 14
strains 15
16
17
18
19
+
3 8
strain
strain
strain
strain strain
strain
l/2a l/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2b l/2b 1/2b 3c 1/2a 1/2a 3a 7 I /2b 1/2a 1/2a 1/2a 1/2c l/2b 1/2b l/2c 1/2a 1/2a 1/2c 1/2b 1/2a 1i2a 3b
human human Reference human human human human human human human food human human Reference Reference human human human human human Reference human Reference human human human human human human Reference
AG 397 LL 93 SLCC 2311 LL 486 GE 1461 ZH 7488 LL 365 AG 7803 AG 7804 SO 8109 FR 2202 ZH 7415, ZH 7416, SLCC 2373 SLCC 2482 BE 7138 ZH 6831 GE 5051 NE 7621 AG 566 SLCC 2755 ZH 5783 SLCC 2372 GE 7620 GE 7434 LL 235 ZH 6692 FR 4383 LL 141 SLCC 2540
monocin
I = 1 = l=NT l=NT I=NT I = I = IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX VIII NT NT
NT NT
Monocin
by serotyping, Serovar
isolates
Origin
of L. nronocytvgenes
Strain
Table 3 Grouping
NT NT NT 1 1 6a 7b NT NT NT NT NT NT NT NY NT NT NT NT NT 2 la la lb 4 6a 6b 7 7a 5
Phage
typing receptor
1 2 3 4 5 6 7 8 8 8 8 8 8 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
type
Combined
B C E NT B4 A3 Cl NT NT NT NT NT NT NT A B3 Cl El F Fl Bl D Dl B3 A2 Al A2 NT A2 NT
methods
phage
phage
typing
Reverse receptor
and three phage monocin
2 3 4 5 6 7 8 8 8 9 10 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Type with all methods except monocinjphage !-,W
Origin
human Reference human human human Reference Reference Poultry human Reference human Reference human human Food human human human human human Reference human human human human human Animal Animal Animal human human
Strain
GE 6075 SLCC 2479 ZH 6290 TI 7006 ZH 6576 SLCC 2316 SLCC 2374 SG 539 LL 458 SLCC 2378 LL 185 SLCC 2315 LL 15, AG 636, BE 1050, BE 6924, LL 124, LL 204, LL 414, LL 21 SLCC 2377 GE 6066 SG 6415, SG 6416, GE 6201 BE 5372 LL 282 LL 283 LL 446 AG 593 GE 3507
Table 3 (contd.)
strain
strain
strain
strain strain
strain
1/2a 3c 1/2b 1/2a 1/2a 4c 4a 4b 4b 4e 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
.__
Serovar
XII XII XII XII XII I = NT I = NT VII X X X X X X X X X X X X X X X X X X X X X X X
Monocin
NT 3 4 6 8 NT 20b 20 NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT
Phage
receptor
;: R S S S S S
39, 39 40 41 42 42 42 42 42
:I
Combined monocin phage type
25 26 27 28 29 30 31 32 33 34 35 35 36 36 36 36 36 36 36 36 37 38
phage
NT B2 A2 NT NT Nl NT L Ll 0 03 03 Pl Pl Pl Pl PI PI PI Pl P2
Reverse receptor
28 29 30 31 32 33 34 35 36 37 38 39 40 40 40 40 40 40 40 41 42 43 44 44 45 46 47 48 49 50 51
Type with all methods except monocin/phage
4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
Environment human human human human human human human human human Food human human human human human human human human Environment Food human human human human human Food human human human human
BE LL ZH LL LL BS ZH LL ZH LL FR BE LL LL LL LL LL GE LL ZH ZH LL LL LL LL ZH BE LL VS LL LL
8637 286 8104 436 457 2392 2945 450 7656 68 1938 7704 191 433 69 471 147 956 168 1925 1198 142 487 180 361 1939 1749 184 2537 105, 140,
Serovar
Origin
Strain
Table 3 (contd.)
X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X
Monocin
12 16 17 19 21 21 12a 14 14 14a 15 15a 15b 15c 16a 16b 16c 17a 17b 17c 17d 18 18 18a 18b 19a 19b 19c 19c 19c 19c
Phage
receptor
M S N 0 NT NT L2 N 02 P3 P 01 NT M2 Rl L2 Pl Ll Ml L L L L L L L L NT N2 N2 N2
Reverse receptor
phage
Combined
monocin
43 44 45 46 41 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 63 64 65 66 67 68 69 69 69
phase type
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 81
Type with all methods except monocin/phage
19c 19c 19c 19c 19c 20a NT 20a
X X X X X X XIII XIII
4b 4b 4b 4b 4b 4b 4b 4b
human human Food Food human Food human human
LL LL BE BE SG PF LL LL
NT, not typeable. “Mother/child pair. bSwiss epidemic type I/ Qolates from same patient. dSwiss epidemic type II.
155d 201, 1004, 1186, 1879 16 460 459
Phage receptor
Monocin
Serovar
Origin
Strain
Table 3 (contd.)
N2 N2 N2 N2 N2 NT S M
Reverse phage receptor
69 69 69 69 69 70 71 72
Combined monocin phage type
81 81 81 81 81 82 83 84
Type with all methods except monocin/phage
E. Bannerman et al. 1 ht. J. Food Microbiology 31 (1996) 245-262
251
number of types and the DI of the monocin/phage combination was the highest among all the methods tested. Only two strains, BE 1050 serovar 4b and reference strain SLCC 2755 serovar 1/2b, had reproducibility problems with both phage methods. The reproducibility of the results was examined by repeating the tests on different occasions at an interval of 4-6 weeks. The readings were identical to the first readings obtained. Probable discrepancies in the phage tests were found to be mainly due to a monocin effect. After leaving the plates overnight at 4°C the differences between lysis caused by the phages and the clear inhibition zone due to monocin could be clearly discerned. The former had plaques of phages accompanied by tailing, while the latter was usually a clear well-defined transparent zone.
4. Discussion Listeriocinogeny has been tried by several workers (Ortel, 1978; Wilhelms and Sandow, 1989; Curtis and Mitchell, 1992) but was not found to be satisfactory for epidemiological typing. The combination of bacteriocinogeny with lysogeny as a tool for epidemiological studies was suggested by Tagg et al. (1976). As far as we know, the first attempt to use this combination on Listeria was carried out by Lebek et al. (1993). Like Curtis and Mitchell (1992), our results clearly show the relationship between serovar and monocin activity. Serogroups l/2, 3 and 7 (group A in this study) strains produced monocins of similar patterns. The serogroup 4 (group B) strains could also be linked together by the similarity of their patterns. There were, however, strains in both groups which were not typeable. The usefulness of monocin typing for epidemiology when taken alone, is questionable since a high proportion of epidemiologically unrelated strains belonged to the same type, but it may be of interest when combined with other methods. In addition, we found as did other workers (Wilhelms and Sandow, 1989; Curtis and Mitchell, 1992; Lebek et al., 1993) that not all L. monocytogenes strains produce monocins. As suggested by Lebek et al. (1993), the possibility of finding other monocin producing types exists. For example, in the present study we identified a monocin class (designated XIII) that was not reported by Lebek et al. (1993). Using the 21 indicator strains, 68% of the 100 strains were typable by the phage receptor analysis under induction with mitomycin C. This figure lies within the range observed by Espaze et al. (1989) and Rocourt et al. (1985) who used the international phage set. Sword and Pickett (1961), however, found only 43 (35%) out of 123 L. monocytogenes strains to be typeable and Tubylewicz (1963a) 3 out of 8. However, in the latter two studies, a different set of indicator strains were used and in addition the phages were induced with ultraviolet light, which is a less effective inducer than mitomycin C (Loessner et al., 1991). Using 17 lysogenic indicator strains for the reverse phage receptor analysis, 80% of the strains were typeable. In comparison, Loessner (1991) typed 86% of 537 L. monocytogenes strains using reverse phage typing. The reverse phage receptor analysis was shown
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4
E. Bunnerman
et al. i ht.
J. Food Microbiology
31 (1996) 245-262
25’)
by the present study to be superior to conventional phage typing. Loessner (1991) demonstrated that the number of phage typeable Listeriu strains could be considerably increased when this method is used. In the present study, only eight of 100 strains tested were not typeable with either of the phage-based methods used. The following seven of these eight non-typeable strains were also non-typeable using the international phage set: two isolates (the mother/baby pair) of serovar 1/2a; of three isolates (three different patients) of serovar 1/2b, two of these patients were in hospital at the same time but in different wards, and one each of serovars 3a (reference strain) and 3c (food) with their own individual profiles for the other methods. When monocin production was combined with phage and reverse phage receptor analysis, all 100 strains could be typed. These seven non-lysogeny typeable strains all produced monocins of type IX. It is possible that these strains were simply not lysogenic, though capable of producing monocins. The two lysogeny tests, the phage receptor and the reverse phage receptor tests, also gave reproducible results, again depending on accurate interpretation of results. The reading of the tests was rendered slightly difficult in a few cases as some strains produced monocins as well as bacteriophages under mitomycin C induction. Using, the phage tailing described by Lebek et al. (1993) as criteria, the results were reproducible. However, induction by mitomycin C for 3 h before filtration might not have allowed sufficient time for slow growing-strains to produce adequate phage titers to enable tailing. There was no obvious correlation between the three methods and the MEE genomic groups of Piffaretti et al. (1989). As seen in Table 3, Type 8 of the combined monocin/phage classification contained strains of both genomic groups of Piffaretti et al. (1989). Serovar 1/2a which belongs to genomic group 2 and serovar 1/2b which belongs to genomic group 1 of Piffaretti et al. (1989) are both found in type 8. A correlation was clearly observed between the serogroups and the monocin typing. There were significant differences between the DIs of the two groups, i.e., serogroup 4 (group B) and the non-serogroup 4 (group A, Table 4). With the exception of RAPD, which has about the same DI for both groups, all the other molecular methods showed higher discrimination indices for the group A, than for the group B strains. However, among the serogroup 4 strains, discrimination was better with phage typing and the two phage methods used in this study. This is an advantage in epidemiological studies, as most of the major epidemics to-date have been due to this serogroup. The comparison of the number of types or subtypes found within the different methods and their respective DIs (Table 4) showed that individually serotyping and monocin typing were the least discriminatory techniques. The number of ribotypes was slightly higher than the number of serotypes, and the DI obtained with the 55 epidemiologically unrelated isolates actually analyzed by ribotyping was between that of serotyping and MEE (Boerlin et al., 1995). The number of types distinguished by MEE and REA was approximately the same, although the discriminatory power of MEE depends on the number of enzymes used (Boerlin et al., 1995). Phage typing and RAPD typing could also distinguish about the same number of types. Phage typing, however, is slightly less discriminatory than RAPD, due to a
260
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
high percentage (32%) of non-typeable strains. This non-typeability could be a drawback when such strains are encountered during an epidemic. Although RAPD was more discriminating than the other methods evaluated, optimal results were only obtained when using two or more primers in combination (Boerlin et al., 1995). The present work shows that monocin production alone is as discriminatory as serotyping, but unlike serotyping, a significant proportion of strains are nontypeable. Phage receptor and the reverse phage receptor methods, however, are more discriminatory, but again both methods have the same drawback as phage typing. In spite of the relatively high number of types obtained with the phage receptor and the reverse phage receptor methods, 32% and 20% of the strains, respectively, were non-typeable, findings similar to those of Loessner (1991). It is conceivable that by extending the range and number of lysogenic indicator strains, a higher percentage of L. monocytogenes strains may become typeable especially with the reverse phage receptor method. The combination of the results obtained by these three methods, however, is as discriminatory or even better than any of the other methods tested (Table 4). This study also showed that the results of the monocin/phage receptor/reverse phage receptor combination correlated well with the results obtained by the other methods. A few additional types could be distinguished by the monocin and phage combination which could not be differentiated by the other methods. In conclusion, our results show that the monocin/phage combination proved reproducible with high discriminatory power and that, in spite of the amount of preparatory work involved, it can be recommended for epidemiological investigations in any laboratory involved in typing Listeriu. For this technique, however, inter-laboratory trials should be carried out and type strains should be available in routine laboratories.
Acknowledgements
We thank Jocelyne Rocourt of the Pasteur Institute, Paris for phage typing our strains, Dorota Nocera, CHUV, Lausanne for ribotyping and Andreas Baumgartner of the Swiss Federal Office for Public Health, Berne for giving us both sets of indicator strains and for his encouragement during the preliminary tests.
References Bille, J. (1990) Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. In: A.J. Miller, J.L. Smith and G.A. Somkuti (editors), Foodborne Listeriosis, Elsevier, Amsterdam, pp. 71-74. Boerlin, P. and Piffaretti, J.-C. (1991) Typing of human, animal, food and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis. Appl. Environ. Microbial. 57, 16241629.
E. Bunnerman et al. /ht. J. Food Microbiology 31 (1996) 245-262
261
Boerlin, P., Bannerman, E., Ischer, F., Rocourt, J. and Bille, J. (1995) Typing Listeriu monocytogenes: a comparison of random amplification of polymorphic DNA with 5 other methods. Res. Microbial. 146, 35-49. Bradley, D.E. and Dewar, C.A. (1966) The structure of phage-like objects associated with non-induced bacteriocinogenic bacteria. J. Gen. Microbial. 45, 399-408. Brosch, R., Chen, J. and Luchansky, J.B. (1994) Pulsed-field fingerprinting of Listeriae: identification of genomic divisions for Listeriu monocytogenes and their correlation with serovar. Appl. Environ. Microbial. 60, 2584-2592. Curtis, G.D.W. and Mitchell, R.G. (1992) Bacteriocin (monocin) interactions among Listeriu monocytogexes strains. Int. J. Food Microbial. 16, 2833292. Espdze, E.P., Gautreau, D., Catimel, B., Miegeville, F., Rocourt, J. and Courtieu, A.L. (1989) An epidemiological survey of human listeriosis in France during 1987, using serotyping and phage typing. Acta Microbial. Hung. 36, 231-234. Graves, L.M., Swaminathan, B., Reeves M.W. and Wenger, J. (1991) Ribosomal DNA fingerprinting of Listeriu monocytogenes using digoxigenin-labeled DNA probe. Eur. J. Epidemiol. 7, 77.-82. Hamon, Y. and Peron, Y. (1962) Etude du pouvoir bacttriocinogene dans le genre Listeria. I. Proprietts gentrales de ces bacteriocines. Ann. Inst. Pasteur. 103, 876-889. Hamon, Y. and Peron, Y. (1963) Etude du pouvoir bacteriocinogene dans le genre Listeriu. II. Individualite et classification des bacteriocines en cause. Ann. Inst. Pasteur 104, 55565. Hunter, P.R. and Gaston, M.A. (1988) Numerical index of the discriminatory ability of typing systems, an application of Simpson’s index of diversity. J. Clin. Microbial. 26, 246552466. Kelstrup, J., Richmond, S., West, C. and Gibbons, J. (1970) Finger-printing human oral streptococci by bacteriocin production and sensitivity. Arch. Oral Biol. 15, II09911 16. Lebek, G., Teysseire, P. and Baumgartner, A. (1993) A method for typing Listeriu monocytogenes strains by classification of listeriocins and phage receptors. Zbl. Bakt. 278, 58-68. Loessner, M.J. (1991) Improved procedure for bacteriophage typing of Listericr strains and evaluation of new phages. Appl. Environ. Microbial. 57, 8822884. Loessner, M.J., Goeppl, S. and Busse, M. (1991) Comparative inducibility of bacteriophage in naturally lysogenic and lysogenized strains of Listeriu spp. by UV light and mitomycin C. Lett. Appl. Microbial. 12, l96- 199. Mazurier, S.-I. and Wernars, K. (1992) Typing of Listeria strains by random amplification of polymorphic DNA. Rev. Microbial. 143, 499-505. Nocera, D., Bannerman, E., Rocourt, J., Jaton-Ggay, K. and Bille, J. (1990) Characterization by DNA restriction endonuclease analysis of Listeriu monocytogenes strains related to the Swiss epidemic of listeriosis. J. Clin. Microbial. 28, 2259-2263. Nocera, D., Altwegg, M., Martinetti-Lucchini, G., Bannerman, E., Ischer, F., Rocourt, J. and Bille, J. (1993) Characterization of Listeriu strains from a foodborne listeriosis outbreak by DNA gene restriction patterns compared to four other typing methods. Eur. J. Clin. Microbial. Infect. Dis. 12, 1622169. Norrung, B. and Gerner-Smidt, P. (1993) Comparison of multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA) and phage typing for typing of Listeria monocytogenes. Epidemiol. Infect. 1I I, 71-79. Ortel, S. (1978) Untersuchungen iiber Monocine. Zbl. Bakt. Hyg. I Abt. Orig. A 242, 72278. Ortel, S. (1989) Listeriocins (monocins). Int. J. Food Microbial. 8, 2499250. Piffaretti, J.-C., Kressebuch, H., Aeschbacheer, M. Bille, J., Bannerman, E., Musser, J.M., Selander, R.K. and Rocourt, J. (1989) Genetic characterization of clones of the bacterium Listeriu monoc,vtogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 85, 3818-3822. Rocourt, J., Audurier, A., Courtieu, A.L., Durst, J., Ortel, S., Schrettenbrunner, A. and Taylor, A.G. (1985) A multi-centre study on the phage typing of Listeriu monocytogenrs. Zbl. Bakt. Hyg. A 259, 489-497. Seeliger, H.P.R. and Hohne, K. (1979) Serotyping of Listeriu monocytogenes and related species, In: T. Bergan and J.R. Norris (editors), Methods in Microbiology, Academic Press, New York, pp. 31-49. Sword, C.P. and Pickett, J. (1961) The isolation and characterization of bacteriophages from Listeriu monocytogenes. J. Gen. Microbial. 25, 241-248.
262
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Tagg, J.R., Dajani, A.S. and Wannamaker, L.W. (1976) Bacteriocins of Gram-positive bacteria. Bact. Rev. 40, 722-756. Tubylewicz, H. (1963a) Studies on the lysogeny of Listerio monocytogenes strains. Bull. Acad. Pal. Sci. (Biol) 11, 5155518. Tubylewicz, H. (1963b) Studies on the bacteriocinogeny of Listeria monocytogenes strains. Bull. Acad. Pol. Sci. (Biol). 11, 519-521. Wesley, I.V. and Ashton, F. (1991) Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics. Appl. Environ. Microbial. 57, 9699996. Wilhelms, D. and Sandow, D. (1989) Preliminary studies on monocine typing of Listeria monocytogenes. Acta Microbial. Hung. 36, 235-238.
Microbiology
Journal
of
31 (1996) 245-262
Typing of Listeria monocytogenes by monocin and
phage receptors Elizabeth Bannerman,
Patrick Boerlin, Jacques Bille*
Centre National de Rkfkence des Listhias, WHO Collaborating Center jbr Foodborne Listeriosis, CHUV, 44, rue du Bugnon, 1001 Lausanne, Switzerland Received
20 July 1995; revised
11 January
1996; accepted
7 February
1996
Abstract One hundred strains of Listeria monocytogenes from both sporadic and epidemic cases were typed by monocin production combined with phage receptor and reverse phage receptor methods. The monocin-phage combination gave 72 types with 100% typability and 97% reproducibility. The results were compared to those of serotyping, phage typing, ribotyping, multilocus enzyme electrophoresis, restriction enzyme analysis and RAPD (random amplification of polymorphic DNA). The monocin/phage types were comparable in terms of discrimination with other methods for epidemiological investigations. The index of discrimination of using the monocin typing and phage receptor/reverse phage receptor method combination (0.99) for both the 87 epidemiologically unrelated strains and the epidemiologically important serogroup 4 strains was the highest of the seven different methods analysed. This combination of methods was simple, highly discriminatory and reproducible and can be carried out in a non-specialized laboratory. However, like most of the other Listeria typing methods, both the method and the indicator test strains need to be standardized. Keywords:
Listeria
monocytogenes;
Monocins;
Phage receptors
1. Introduction Listeriocins * Corresponding
0168-1605/96/$15.00
(Tubylewicz, author:
1963a,b)
Tel: 41 21 3144057,
0 1996 Elsevier
PII SO168-1605(96)01003-3
or monocins
Science
(Hamon
fax: 41 21 3144060.
B.V. All rights
reserved
and P&on,
1962, 1963)
246
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
are bacteriocins of the genus Listeria and were first described by Sword and Pickett (1961). Monocins are resistant to trypsin, and are inactivated at 50°C and at pH 4.5. Monocins have been described as defective phages without heads (Bradley and Dewar, 1966) with antibiotic-type effects against Listeria and other Gram-positive organisms (Ortel, 1989). The monocins with this bactericidal effect are thus comparable to other bacteriocins such as colicins, pyocins, or staphylococcins. Bacteriocins of genera other than Listeria have found application in epidemiological studies as specific markers for bacteria (Tagg et al., 1976). Typing schemes have been established based on either the production of, or the sensitivity to a range of different bacteriocins. Hamon and P&on (1963) found 10 different types of monocins and Tubylewicz (1963b) classified 4 types, both after ultra-violet (UV) induction of Listeria strains. Ortel (1978) described 6 different monocins after mitomycin C induction of L. monocytogenes strains. Tagg et al. (1976) suggested that greater discrimination for typing may be obtained by the combination of bacteriocin production with bacteriocin sensitivity, or by either or both bacteriocin-based methods used with other typing methods, such as serotyping or phage typing. The combination of bacteriocin production and sensitivity was useful for typing streptococci (Kelstrup et al., 1970) but was found not to be discriminatory for subtyping Listeria (Curtis and Mitchell, 1992). The combination of bacteriocin typing with serotyping of Listeriu was also poorly discriminatory (Wesley and Ashton, 1991). Recently, Lebek et al. (1993) combined phage typing (phage receptor and reverse phage receptor analysis) with the monocinogeny and demonstrated a relatively high discriminatory power among some Listeria strains. Among the typing methods available for L. monocytogenes, serotyping (Seeliger and Hiihne, 1979) and phage typing (Rocourt et al., 1985) are relatively simple and cost-effective. Serotyping, however is not discriminatory enough for epidemiological studies, while phage typing, though discriminating, is limited because not all Listeriu strains are phage typable (Rocourt et al., 1985; Espaze et al., 1989). Multilocus enzyme electrophoresis (MEE) is discriminatory (Boerlin and Piffaretti, 1991) and also allows estimation of genomic relationships among strains (Piffaretti et al., 1989). This method however, is time-consuming and labour intensive. Restriction enzyme analysis (REA) is easy to perform and discriminatory (Nocera et al., 1990; Wesley and Ashton, 1991; Nsrrung and Gerner-Smidt, 1993) but the electrophoretic patterns obtained are often difficult to interpret and like the MEE needs electrophoresis equipment. Ribotyping has also been successfully used to type L. monocytogenes (Graves et al., 1991; Nocera et al., 1993) but this method is also labour intensive and not discriminatory enough, particularly for the clinically significant serovar 4b strains (Nocera et al., 1993). The most recent typing method is the random amplified polymorphic DNA (RAPD) analysis (Mazurier and Wernars, 1992) which seems to be highly discriminatory (Boerlin et al., 1995). Like the other molecular methods, it needs special equipment and reproducibility problems may sometimes be encountered (Boerlin et al., 1995). Another molecular
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
241
typing method currently used but not evaluated here, is pulsed-field gel electrophoresis (PFGE). Brosch et al. (1994) demonstrated that PFGE can also be used to divide Listeria into genomic groups which in turn can be used to correlate serotypes. Listeriocinogeny combined with phage receptor and reverse phage receptor analyses, though time consuming, can be carried out in any laboratory and does not need special equipment. In an attempt to determine whether in a case of an epidemic, this combination of techniques could replace any of the other more cumbersome methods in a non-specialized laboratory, we compared the approach of phage typing in combination with bacteriocin typing with 6 other Listeria typing methods for discriminatory power and for utility in epidemiological studies.
2. Materials and methods 2.1. Bacterial strains
The same set of strains used by Boerlin et al. (1995) and consisting of 100 isolates of serovars 1/2a (19) 1/2b (8) 1/2c (3) 3a (1) 3b (1) 3c (2) 4a (1) 4b (61) 4c (I), 4d (1) 4e (1) and 7 (1) were used in the present study. Except for phage typing which was done in Paris (Rocourt et al., 1985) all strains have been previously typed in our laboratory (Boerlin et al., 1995). With the exception of the 12 L. monocytogenes serotyping reference strains from the Special Listeria Culture Collection (SLCC; Wiirzburg, Germany), the strains selected were isolates from human (75) animal (3) food (8) and environmental (2) origin. Serovar 4b strains which are frequently encountered in human listeriosis and which are less distinguishable by other methods are particularly well represented in this study. Serovar 4b strains analyzed included 7 phage type I and 6 phage type II isolates from the Swiss epidemic of 1983-1987 (Bille, 1990). Also included were epidemiologically related mother/baby isolates and isolates from different sites of the same patient. All indicator strains (7 isolates for monocin testing and 21 isolates for phage testing; Lebek et al., 1993) were obtained from the Swiss Federal Office for Public Health. Berne. 2.2. Typing methods 2.2.1. Monocin analyses
Monocin typing was carried out using the technique described by Lebek et al. (1993) but with 5 instead of 14 spots per Petri dish (8.5 cm diameter). 2.2.2. Phage receptor analysis
The multiplication described by Lebek eleven indicator (lstrains for serogroup
of bacteriophages in Listeria was induced by mitomycin C, as et al. (1993). All the strains were subjected to this test using 11) strains for serogroups l/2 and 3 and ten (12-21) indicator 4. L. monocytogenes serovar 7 which does not belong to either
248
E. Bannerman et al. /ht.
J. Food Microbiology 31 (1996) 245-262
group, was tested with both sets of indicator strains. It was important that the flooding of the plates, as well as the subsequent decanting was unidirectional 2.2.3. Reverse phage receptor analysis The reverse phage receptor analysis was carried out on all 100 strains. Using the same technique described for the phage receptor analysis, the strains to be typed were taken as indicator strains and the lysogenic strains (1 to 8 for serogroups l/2 and 3; 12 to 20 for serogroup 4) served as the test strains (Lebek et al., 1993). 2.3. Reading of the tests All the plates were read against a dark background or with an oblique illuminating apparatus. For monocin tests, this enabled the zones of inhibition and for the phage tests, the presence of phage plaques to be clearly seen. For the phage tests, reactions were considered positive when plaques of phages (usually with tailing) were seen. Clear well-defined zones were considered negative. The results were always confirmed by a second reading after the plates were stored overnight at 4°C. 2.4. Reproducibility tests All 100 strains were typed at least twice. The tests were all repeated after 4-6 weeks. Strains with discrepancies were subjected to two additional analyses; the fourth test was for confirmation. For the two phage tests, all non typable strains, the epidemiologically related strains and L. monocytogenes serovar 7 were also tested three times with both sets of indicator strains. 2.5. Index of discrimination (DI)
The index of discrimination Hunter and Gaston (1988).
was calculated
using the formula
proposed
by
3. Results 3.1. Monocin typing
The identification of the different types of monocins (Table 1) was based on that of Lebek et al. (1993) for listeriocin testing. Three different zones of inhibition (large transparent, small transparent and large diffuse) were observed. As a result of their monocin reactions, all the L. monocytogenes non-serogroup 4 strains, that is, L. monocytogenes serogroups l/2, 3 and 7 will be referred to as group A strains and the serogroup 4 (serovars 4a, 4b, 4c, 4d, 4e) as group B strains. Four monocin types (I, VIII, IX, and XII) were found among the 35 isolates of the group A strains with 22 (62.8%) strains belonging to type IX. Seven strains of this group were not typeable with the 7 indicator strains used in the monocin test (Type I = NT).
I = NT VII X XIII I = NT VIII IX XII
Serogroup 4(65 strains)
Non-serogroup
NT, not typeabie. -, resistant (no zone of inhibition). 1, large and transparent. 2, large and diffuse. 3, small and transparent.
4(35 strains)
Monocin type
L. monocytogenes
Table 1 Identification of the monocin types
7 1 22 5
2 1 60 2
No. of strains
1 1 1
1 1
a
3
2 2 2
b
3 3 3
C
2 2
d
e
f
Susceptibility of indicator strains to different monocins g
2
F
250
E. Bannerman et al. / ht. J. Food Microbiology 31 (1996) 245-262
However, all 7 strains were typeable by either the phage receptor or the reverse phage receptor methods. Reference strain, serovar 3b (SLCC 2540) was the only strain which showed reproducibility problems with the monocin test. Two additional tests, however, gave the same readings as the first test and the strain was eventually identified as a type VIII. Four different monocin-types (I VII, X and XIII) were found among the 65 isolates of the group B strains with 60 strains (92%) belonging to type X. Two reference strains, SLCC 2374 (serovar 4a) and SLCC 2376 (serovar 4c) did not produce monocins and were assigned to type I. All the results were reproducible for this group. The reproducibility for monocin typing was thus 99% and the overall typeability was 91%. 3.2. Phage typing The scheme used for interpreting the phage analysis tests is shown in Table 2a (serogroups other than 4) and Table 2b (serogroup 4). The criteria of Lebek et al. (1993) were used for reading of the plates. Of the group A strains, 18 (51.4%) and 22 (62.8%) out of 35 strains were typeable with the phage receptor and the reverse phage receptor tests, respectively. The 65 group B strains (Table 2b), gave 40 (61.5%) and 58 (89.2%) typeable strains, respectively, with the phage receptor and the reverse phage receptor tests with the set of indicator strains used. Reproducibility problems were encountered with the same two strains (BE 1050 and SLCC 2755) for both tests, leading to an overall reproducibility of 98% for both the phage receptor and the reverse phage receptor methods. Among the 35 group A strains, 15 different types were identified with the phage receptor test while 30 different types were found among the 65 group B strains. The reverse phage receptor analysis gave 17 and 26 different types for groups A and B, respectively. 3.3. Combined results When the three methods were combined, 72 different types were obtained (Table 3) and the overall typability was lOO%, with 97% reproducibility. The seven isolates belonging to the Swiss epidemic type I strains (sv 4b, ribovar I, ET 1, REA-A, lysovar 47/108/340/2389/2425/2671/3274 and RAPD type 2) were indistiguinshable by the three methods (type 36, Table 3), as well as by the six other methods. Likewise, the six Swiss epidemic type II isolates comprised a single type using the three methods (type 69, Table 3). However, the Swiss type I strains (type 36) were differentiated from the Swiss type II strains (type 69) by the combined monocin/phage methods. Two isolates from the same patient but obtained from different sites (SG 6415 = blood and SG 6416 = ascites; type 39, Table 3) could also be paired together using the present three methods in combination with the six other methods, previously described. The same could be done with a mother/ child pair of isolates (ZH 7415 and ZH 7416; type 8, Table 3). For the 87 epidemiologically unrelated strains, the number of types and the index of discrimination (DI) obtained with each method are shown in Table 4. The
+ + +
+ t
+ +
+
+
+ + + t
+ + t +
+
6 6 6 7 7 I 8
6 6a 6b 7 la 7b 8
+ t + t + + +
+
t
5
5
+ t + t + + +
+
+
+ + + + + + +
+
+
+
+
+
+
H
G
NT A Al A2 A3 B Bl 82 B3 84 C Cl D Dl E El F FI
4
11
4
10
+
9
+
8
3
7
3
6
2
5
2
4
0 1 1 1
3
NT I la lb
2
Code
strains
No. of plaques
Code
Indicator
4 phage types
Reverse
4 phage types
4 and (b) serogroup
Phage receptors
non-serogroup
(a) non-serogroup
for interpreting
for interpreting
(a) Diagram
Table 2 Diagram
8
I
6 6
5 5
3 3 4 4
2 2 2 2 2
0 1 I 1
No. of plaques
phage receptors
+
+
+
+
t
+ +
+
I -
t
+
+ +
+ + +
t + +
+
3
strains
+ + f
t + +
+
+
2
Indicator
+
4
+
t
+ + + +
+
+
+
+
+
5
6
+
+
+ + + + + +
+ + +
+
7
+
+
+ + + + +
+
+
+
8
for interpreting
2
3 3
4 4 4 4 5 5 5 5 6 6 6 6 6 1 7 I 8 8 8 8 9 9 9 10
13
14 14a
15 15a 15b 15c 16 16a 16b 16~. 17 17a 17b 17c 17d 18 18a 18b 19 19a 19b 19c 20 20a 20b 21
reaction.
0
NT 12 12a
+ , positive
No. of plaques
Code
Phage recrptors
(b) Diagram
Table 2
f
+ +
+ + +
+
+ + + +
+ +
+ +
12
+ + + + + + + t + +
+
+ +
13
Indicator
serogroup
+ + + + + + t +
+ + + + +
+
+ + +
+
14
strains
+ + + +
+ + +
+
+ + + +
+ + + + + + +
+
15
4 phage types
+ + + + +
+ + + + +
+ +
+ +
+ +
+ +
+
16
+ + + + + + + + + + t +
+
+
17
+ +
+ +
+ +
+ +
+
+
+
18
19
+ + + + + + +
+ + +
+ + +
+ +
i + +
+
20
-
+ + + + + + + + + + + + +
+ + + + + + + t + +
+ +
+
21
T
s
R RI
42
z1
NT L LI L2 M Ml M2 N Nl N2 0 01 02 03 P Pl P2 P3
Code
Reverse
9
0 1 1 1 2 2 2 3 3 3 4 4 4 4 5 5 5 5 6 6 6
No. of plaques
phage receptors
+
+
+ +
+ +
+ + +
+ +
+
12
i-
+
+ +
+ + + + + + +
+
+
13
Indicator 14
strains 15
16
17
18
19
+
3 8
strain
strain
strain
strain strain
strain
l/2a l/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2b l/2b 1/2b 3c 1/2a 1/2a 3a 7 I /2b 1/2a 1/2a 1/2a 1/2c l/2b 1/2b l/2c 1/2a 1/2a 1/2c 1/2b 1/2a 1i2a 3b
human human Reference human human human human human human human food human human Reference Reference human human human human human Reference human Reference human human human human human human Reference
AG 397 LL 93 SLCC 2311 LL 486 GE 1461 ZH 7488 LL 365 AG 7803 AG 7804 SO 8109 FR 2202 ZH 7415, ZH 7416, SLCC 2373 SLCC 2482 BE 7138 ZH 6831 GE 5051 NE 7621 AG 566 SLCC 2755 ZH 5783 SLCC 2372 GE 7620 GE 7434 LL 235 ZH 6692 FR 4383 LL 141 SLCC 2540
monocin
I = 1 = l=NT l=NT I=NT I = I = IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX IX VIII NT NT
NT NT
Monocin
by serotyping, Serovar
isolates
Origin
of L. nronocytvgenes
Strain
Table 3 Grouping
NT NT NT 1 1 6a 7b NT NT NT NT NT NT NT NY NT NT NT NT NT 2 la la lb 4 6a 6b 7 7a 5
Phage
typing receptor
1 2 3 4 5 6 7 8 8 8 8 8 8 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
type
Combined
B C E NT B4 A3 Cl NT NT NT NT NT NT NT A B3 Cl El F Fl Bl D Dl B3 A2 Al A2 NT A2 NT
methods
phage
phage
typing
Reverse receptor
and three phage monocin
2 3 4 5 6 7 8 8 8 9 10 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Type with all methods except monocinjphage !-,W
Origin
human Reference human human human Reference Reference Poultry human Reference human Reference human human Food human human human human human Reference human human human human human Animal Animal Animal human human
Strain
GE 6075 SLCC 2479 ZH 6290 TI 7006 ZH 6576 SLCC 2316 SLCC 2374 SG 539 LL 458 SLCC 2378 LL 185 SLCC 2315 LL 15, AG 636, BE 1050, BE 6924, LL 124, LL 204, LL 414, LL 21 SLCC 2377 GE 6066 SG 6415, SG 6416, GE 6201 BE 5372 LL 282 LL 283 LL 446 AG 593 GE 3507
Table 3 (contd.)
strain
strain
strain
strain strain
strain
1/2a 3c 1/2b 1/2a 1/2a 4c 4a 4b 4b 4e 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
.__
Serovar
XII XII XII XII XII I = NT I = NT VII X X X X X X X X X X X X X X X X X X X X X X X
Monocin
NT 3 4 6 8 NT 20b 20 NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT
Phage
receptor
;: R S S S S S
39, 39 40 41 42 42 42 42 42
:I
Combined monocin phage type
25 26 27 28 29 30 31 32 33 34 35 35 36 36 36 36 36 36 36 36 37 38
phage
NT B2 A2 NT NT Nl NT L Ll 0 03 03 Pl Pl Pl Pl PI PI PI Pl P2
Reverse receptor
28 29 30 31 32 33 34 35 36 37 38 39 40 40 40 40 40 40 40 41 42 43 44 44 45 46 47 48 49 50 51
Type with all methods except monocin/phage
4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b
Environment human human human human human human human human human Food human human human human human human human human Environment Food human human human human human Food human human human human
BE LL ZH LL LL BS ZH LL ZH LL FR BE LL LL LL LL LL GE LL ZH ZH LL LL LL LL ZH BE LL VS LL LL
8637 286 8104 436 457 2392 2945 450 7656 68 1938 7704 191 433 69 471 147 956 168 1925 1198 142 487 180 361 1939 1749 184 2537 105, 140,
Serovar
Origin
Strain
Table 3 (contd.)
X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X
Monocin
12 16 17 19 21 21 12a 14 14 14a 15 15a 15b 15c 16a 16b 16c 17a 17b 17c 17d 18 18 18a 18b 19a 19b 19c 19c 19c 19c
Phage
receptor
M S N 0 NT NT L2 N 02 P3 P 01 NT M2 Rl L2 Pl Ll Ml L L L L L L L L NT N2 N2 N2
Reverse receptor
phage
Combined
monocin
43 44 45 46 41 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 63 64 65 66 67 68 69 69 69
phase type
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 81
Type with all methods except monocin/phage
19c 19c 19c 19c 19c 20a NT 20a
X X X X X X XIII XIII
4b 4b 4b 4b 4b 4b 4b 4b
human human Food Food human Food human human
LL LL BE BE SG PF LL LL
NT, not typeable. “Mother/child pair. bSwiss epidemic type I/ Qolates from same patient. dSwiss epidemic type II.
155d 201, 1004, 1186, 1879 16 460 459
Phage receptor
Monocin
Serovar
Origin
Strain
Table 3 (contd.)
N2 N2 N2 N2 N2 NT S M
Reverse phage receptor
69 69 69 69 69 70 71 72
Combined monocin phage type
81 81 81 81 81 82 83 84
Type with all methods except monocin/phage
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number of types and the DI of the monocin/phage combination was the highest among all the methods tested. Only two strains, BE 1050 serovar 4b and reference strain SLCC 2755 serovar 1/2b, had reproducibility problems with both phage methods. The reproducibility of the results was examined by repeating the tests on different occasions at an interval of 4-6 weeks. The readings were identical to the first readings obtained. Probable discrepancies in the phage tests were found to be mainly due to a monocin effect. After leaving the plates overnight at 4°C the differences between lysis caused by the phages and the clear inhibition zone due to monocin could be clearly discerned. The former had plaques of phages accompanied by tailing, while the latter was usually a clear well-defined transparent zone.
4. Discussion Listeriocinogeny has been tried by several workers (Ortel, 1978; Wilhelms and Sandow, 1989; Curtis and Mitchell, 1992) but was not found to be satisfactory for epidemiological typing. The combination of bacteriocinogeny with lysogeny as a tool for epidemiological studies was suggested by Tagg et al. (1976). As far as we know, the first attempt to use this combination on Listeria was carried out by Lebek et al. (1993). Like Curtis and Mitchell (1992), our results clearly show the relationship between serovar and monocin activity. Serogroups l/2, 3 and 7 (group A in this study) strains produced monocins of similar patterns. The serogroup 4 (group B) strains could also be linked together by the similarity of their patterns. There were, however, strains in both groups which were not typeable. The usefulness of monocin typing for epidemiology when taken alone, is questionable since a high proportion of epidemiologically unrelated strains belonged to the same type, but it may be of interest when combined with other methods. In addition, we found as did other workers (Wilhelms and Sandow, 1989; Curtis and Mitchell, 1992; Lebek et al., 1993) that not all L. monocytogenes strains produce monocins. As suggested by Lebek et al. (1993), the possibility of finding other monocin producing types exists. For example, in the present study we identified a monocin class (designated XIII) that was not reported by Lebek et al. (1993). Using the 21 indicator strains, 68% of the 100 strains were typable by the phage receptor analysis under induction with mitomycin C. This figure lies within the range observed by Espaze et al. (1989) and Rocourt et al. (1985) who used the international phage set. Sword and Pickett (1961), however, found only 43 (35%) out of 123 L. monocytogenes strains to be typeable and Tubylewicz (1963a) 3 out of 8. However, in the latter two studies, a different set of indicator strains were used and in addition the phages were induced with ultraviolet light, which is a less effective inducer than mitomycin C (Loessner et al., 1991). Using 17 lysogenic indicator strains for the reverse phage receptor analysis, 80% of the strains were typeable. In comparison, Loessner (1991) typed 86% of 537 L. monocytogenes strains using reverse phage typing. The reverse phage receptor analysis was shown
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by the present study to be superior to conventional phage typing. Loessner (1991) demonstrated that the number of phage typeable Listeriu strains could be considerably increased when this method is used. In the present study, only eight of 100 strains tested were not typeable with either of the phage-based methods used. The following seven of these eight non-typeable strains were also non-typeable using the international phage set: two isolates (the mother/baby pair) of serovar 1/2a; of three isolates (three different patients) of serovar 1/2b, two of these patients were in hospital at the same time but in different wards, and one each of serovars 3a (reference strain) and 3c (food) with their own individual profiles for the other methods. When monocin production was combined with phage and reverse phage receptor analysis, all 100 strains could be typed. These seven non-lysogeny typeable strains all produced monocins of type IX. It is possible that these strains were simply not lysogenic, though capable of producing monocins. The two lysogeny tests, the phage receptor and the reverse phage receptor tests, also gave reproducible results, again depending on accurate interpretation of results. The reading of the tests was rendered slightly difficult in a few cases as some strains produced monocins as well as bacteriophages under mitomycin C induction. Using, the phage tailing described by Lebek et al. (1993) as criteria, the results were reproducible. However, induction by mitomycin C for 3 h before filtration might not have allowed sufficient time for slow growing-strains to produce adequate phage titers to enable tailing. There was no obvious correlation between the three methods and the MEE genomic groups of Piffaretti et al. (1989). As seen in Table 3, Type 8 of the combined monocin/phage classification contained strains of both genomic groups of Piffaretti et al. (1989). Serovar 1/2a which belongs to genomic group 2 and serovar 1/2b which belongs to genomic group 1 of Piffaretti et al. (1989) are both found in type 8. A correlation was clearly observed between the serogroups and the monocin typing. There were significant differences between the DIs of the two groups, i.e., serogroup 4 (group B) and the non-serogroup 4 (group A, Table 4). With the exception of RAPD, which has about the same DI for both groups, all the other molecular methods showed higher discrimination indices for the group A, than for the group B strains. However, among the serogroup 4 strains, discrimination was better with phage typing and the two phage methods used in this study. This is an advantage in epidemiological studies, as most of the major epidemics to-date have been due to this serogroup. The comparison of the number of types or subtypes found within the different methods and their respective DIs (Table 4) showed that individually serotyping and monocin typing were the least discriminatory techniques. The number of ribotypes was slightly higher than the number of serotypes, and the DI obtained with the 55 epidemiologically unrelated isolates actually analyzed by ribotyping was between that of serotyping and MEE (Boerlin et al., 1995). The number of types distinguished by MEE and REA was approximately the same, although the discriminatory power of MEE depends on the number of enzymes used (Boerlin et al., 1995). Phage typing and RAPD typing could also distinguish about the same number of types. Phage typing, however, is slightly less discriminatory than RAPD, due to a
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high percentage (32%) of non-typeable strains. This non-typeability could be a drawback when such strains are encountered during an epidemic. Although RAPD was more discriminating than the other methods evaluated, optimal results were only obtained when using two or more primers in combination (Boerlin et al., 1995). The present work shows that monocin production alone is as discriminatory as serotyping, but unlike serotyping, a significant proportion of strains are nontypeable. Phage receptor and the reverse phage receptor methods, however, are more discriminatory, but again both methods have the same drawback as phage typing. In spite of the relatively high number of types obtained with the phage receptor and the reverse phage receptor methods, 32% and 20% of the strains, respectively, were non-typeable, findings similar to those of Loessner (1991). It is conceivable that by extending the range and number of lysogenic indicator strains, a higher percentage of L. monocytogenes strains may become typeable especially with the reverse phage receptor method. The combination of the results obtained by these three methods, however, is as discriminatory or even better than any of the other methods tested (Table 4). This study also showed that the results of the monocin/phage receptor/reverse phage receptor combination correlated well with the results obtained by the other methods. A few additional types could be distinguished by the monocin and phage combination which could not be differentiated by the other methods. In conclusion, our results show that the monocin/phage combination proved reproducible with high discriminatory power and that, in spite of the amount of preparatory work involved, it can be recommended for epidemiological investigations in any laboratory involved in typing Listeriu. For this technique, however, inter-laboratory trials should be carried out and type strains should be available in routine laboratories.
Acknowledgements
We thank Jocelyne Rocourt of the Pasteur Institute, Paris for phage typing our strains, Dorota Nocera, CHUV, Lausanne for ribotyping and Andreas Baumgartner of the Swiss Federal Office for Public Health, Berne for giving us both sets of indicator strains and for his encouragement during the preliminary tests.
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