International Journal of Fotnl Microbiology. 15 (1992) 347-356 ~ 1','92 Elsevier Science Publishers B.V. All rights reserved 016.~-16l)5/92/$1)5.011

347

FOOD IX}476

Restriction fragment length polymorphism analysis of Listeria monocytogenes and its application to epidemiological investigations A.E. Lew and P.M. Desmarchelier Tropical Ih'alth Program ,;zd Department of Microhiology. Umccr.~io"o ft2ueenshmd, Brisb, nc, ()lu't.tlxhmc~ Att.~tralia

(Received 19 June 1991; accepted 25 February 1992)

The restriction fragment length I~)lymorphisms (RFLPs) of 64 random and potentially related strains of Listcria ~,,,.,n~w~'togent,.~"were analysed and compared using a probe comprised of two L, nlowzot'ylogt'nt's chromosome fragments cloned into a A vector. Twelve RFLP types were defined using 14 isolates of clinical origin, 42 food isolates and eight food associated environmental strains. Of the RFLP types, some were common to a particular serovar and source, whereas others were widespread amongst all serovars and sources. One of the two most common RFLP patterns was associated with serovar ! / 2 isolates from ft~d or the environment, whereas another dominant pattern was associated most commonly with serovar four isolates from all sources. The potential relationships between epidem~ologically related strains were examined, with the analysis of types from a suspected listeriosis outbreak, from clinical maternal-fi~ctal cases, and from an ice-cream factory environmental study. Serotyping alone was not a sufficient marker for the comparison of these strains whereas further discrimination of strains was possible with RFLP analysis. Key words: Li.~'teriamonotTtogt'nes: RFLP: Epidemiology

Introduction T y p i n g m e t h o d s u s e d to d i s t i n g u i s h b e t w e e n s t r a i n s o f Listeria montn'ytvgen~'x have t r a d i t i o n a l l y i n c l u d e d s e r o t y p i n g a n d b a c t e r i o p h a g e typing. S e r o t y p i n g o f L. m o n o c y t o g e n e s is b a s e d o n t h e a n a l y s i s o f s o m a t i c a n d f l a g e l l a r a n t i g e n s , c~,abinat i o n s o f w h i c h h a v e d e f i n e d 13 d i f f e r e n t s c r o v a r s o f t h e o r g a n i s m ( S e e l i g e r a n d H o h n e , 1979). T h e m a j o r i t y o f c a s e s o f l i s t e r i o s i s a r e c a u s e d by s e r o v a r s 1 / 2 a , 1 / 2 b a n d 4b ( M c L a u c h l i n , 1987), c o n s e q u e n t l y t h e d i s c r i m i n a t i o n o f s e r o t y p i n g in e p i d e m i o l o g i c a l s t u d i e s c a n b e r e s t r i c t i v e . P h a g e t y p i n g is m o r e d i s c r i m i n a t i v e b u t n o t all s t r a i n s a r e t y p a b l e by this s c h e m e ( R o c o u r t et al., 1985). L o e s s n e r a n d his

Correslnmdence address: P.M. Desmarchelier, Tropical Health Program, Unive~ity of Queensland. Brisbane, Queensland, Australia.

348 co-workers (1991) have improved the overall typability of Listeria spp. using an extended set of bacteriophages in a reversed phage typing system. Alternative methods for typing epidemiologically related strains of L. monocytogenes have been investigated. Piasmid profiles (Fistrovici and Collins-Thompson, 1990), restriction endonuclease digests (Nocera et al., 1990; Szabo and Desmarchelier, 1990; Wesley and Ashton, 1991), multilocus enzyme electrophoresis (MEE) (Bibb et al., 1990) and DNA probes (Notermans et al., 1989) have been used to discriminate isolates of L. monotTtogenes. The use of plasmid profiles to monitor L. monocytogenes has been found to be of limited use as a typing scheme due to the observed lack of plasmids in this species (Fistrovici and Collins-Thompson, 1990). The use of DNA restriction enzyme fragment analyses for the typing of L. monocytogenes strains has proved to be very discriminative (Ridgway and Brown, 1989; Nocera et al., 1990; Szabo and Desmarchelier, 1990; Wesley and Ashton, 1991); however, a problem arises in the comparison of large numbers of restriction fragment profiles due to the complexity of the patterns. This problem can be simplified by the use of probes which selectively hybridise with a small fraction of the restriction fragments (Wesley et al., 1990; Owen, 1989; Knight et al., 1990). Probes consisting of random L. monocytogenes sequences in a phage lambda vector have been used to analyse inter-strain RFLP's of L. monocytogenes isolates (Saunders et al., 1989). We describe here the evaluation of these probes to discriminate Australian L. monocytogenes isolates, and their potential use in epidemiological investigations.

Methods and materials

Bacterial strains attd preparation of chromosomal DNA Sixty-four isolates with relevant epidemiologieal data (when available) were obtained from other laboratories for the inclusion in this study. The strains included 14 clinical isolates, 42 food isolates and eight isolates from environments associated with some of the food isolates. The majority of these isolates were from random sources. The potentially epidemiologically related strains were donated from unpublished studies and included strains isolated during a suspected outbreak of listeriosis, four pairs of clinical maternal-foetal isolates, and isolates from ice-cream and from the associated factory processing environment. Isolates of L. monocytogenes were identified on the basis of biochemical and serological reactions and were cultured on tryptone z~,i:~cose yeast extract agar as published previously (Szabo and Desmarchelier ~990). Total chromosomal DNA was extracted following the method of Saunclers et al. (1988) and DNA yields, usually ranging from 30 to 200 tzg, were determined spectrophotometrically. Restriction endonuclease digestion and electrophoresis Five ~g of each bacterial DNA was digested by 8 units of Nci 1 (BRL, Bethesda Research Laboratories, Inc., Gaithersburg, MD, USA) for 18 h in 50 ~1 solution using the buffer supplied by the manufacturer. The restriction fragments were

349 separated by agarose gel electrophoresis in ll.Sc~ (w/v) agarose in Tris-acetate buffer (40 mM Tris, 20 mM acetate, 41} mM EDTA) at i.2 Vcm-I overnight and then transferred to Nylon (Hybond-N, Amersham International, Amersham, England) filters by the method of Southern (1975). A mixture of restriction fragments comprising Pstl and EcoRl digests of A phage DNA were run as molecular weight standards.

Labelling and hybrMisation of probes Probes ANS32 and ANS35"and purified probe DNAs were supplied by Dr. N. Saunders of the CPH Laboratory, London, UK. Probe DNAs were prepared from the lambda phage clones as described previously (Saunders et al., 1989). The probes were labelled with biotin-li-dUTP using the BRL Nick Translation Kit according to the manufacturer's instructions (BRL). Southern blots were prehybridised for 3 h and then hybridised for 18 h at 42°C in hybridisation solution which has been described previously (Saunders et al., 1988). Following hybridisation the filters were washed at low stringency in 2 x SSC, 0.1% SDS at 50°C (2 × 15 min a~d I x 31) min) and were developed using the Blu-gene biotin detection reagents (BRL) according to the manufacturer's instructions.

DNA fi'agment size dewrmbmtion and comparison Fragment sizes were calculated from migration distances using the A standards and a BASIC computer program (exlin2) which uses a double exponential curve fitting algorithm (kindly supplied by Mr A. Healey of the Queensland Institute of Medical Research, The Bancroft Centre, 31H) Herston Road, Brisbane, Queensland, Australia). Normalised migration values were obtained from fragment sizes by using a formula relating migration to size (irino et al., 1988). The resulting patterns were grouped into types representing common RFLP groups, in addition, representative strains of the Australian RFLP patterns were sent to Dr N. Saunders (Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory (CPH), 61 Colindale Avenue, London, NW9 5HT, UK) for comparison and confirmation of results.

Results

Dicersity o1" RFLP types The 64 strains of L. monoo'togenes from various related and unrelated Australian sources were typed into 12 RFLP groups representing 12 different patterns of molecular weight bands. A summary of RFLP types which were observed are presented in Table !. Seven RFLP types were observed amongst the 42 food isolates, five major types were found among clinical strains, and three RFLP types were observed amongst the eight environmental strains. Some pattern(s) were unique to strains from a particular source (food, clinical, environmental), whereas one pattern (RFLP type 6) was commonly found in all sources. Figure 1 gives a normalised representation of all of the RFLP patterns.

35O TABLE I Summary of L. monocytogenes RFLP types Source

Ser(war

RFLP types

Fot)d (n " = 42)

I/2 3 4

5(I) h 6(2). 8(5), tXI4), iIXI) 6(3). ¢XI), 12(i). 5[2) 6(71, 7(4), (XI )

E n v i r o n m e n t ( , = 8)

I/2 4 I/2

9( I ). 1 !( I ) 6(6) 1(2). 2(I). 3(2). 4(21 6(71

I l u m a n (n = 14)

4

"n" denotes the ilunlber o f isolates. (n) h represents the numher of isolates fl)r each RFLP lype of each serovar and source. "

RFLP type 9 was not h)und among clinical strains but was relatively common in foods and most commonly associated with the serovar 1/2 strains, in contrast, RFLP type 6 included strains of all sercwars but was most commonly associated with the strains of serovar 4. An example of these two dominant RFLP types is shown in Fig. 2.

RFLP p a t t e r n 1

2

!

l

3 ,

4 i

5

6

~

7

i

i

8

9

i

i

10

11

12

'

'

'

. . . . . . . .

Kb 30 20 10

m

~

m

5 4

2

B

m 1

0.5

Fig. 1. Normaliscd graph of L. monoc.vtogtvzes RFLP types showing the migration patterns of the DNA restriction fragments after cleavage by Nci I and hybridisation to the lambda probes. Dotted lines denote faint hands.

351

b~

-,,-

7 421

1

5

1

4 749

1

3

530

,1

2

838

-.---" --.

2 459 2 146 1 986 1 700

.., "

a

226 057 501

21 14 11

1

304

1 1

159 093

--

805

-:

514 448

b

Fig. 2. Nci I digests or 13stcria monen'ytogcm',~ chromosonlal DNA southern hybridizations to biotinlabelled lamhda probes. Lane a, RFLP type 9: lane h, RFLP type 6.

T h e serovar 4 isolates (n = 25) wcrc dividcd into 3 R F L P types and were the least diverse group. Sertwar 1 / 2 isolates (n = 32) were found to be more heterogeneous with l0 R F L P types observed. Serowtr 3 isolates (n = 7) were diversified further by R F L P analysis where four R F L P types were observed. Representative strains of R F L P types 2, 3, 4, 5, 6, 7, 9 and 12 were typed by the Central Public Health' ( C P H ) laboratory in London, UK. These Australian types were indistinguishable from types already isolated at that laboratory (personal communication). In addition, the C P H laboratory's pattern equivalent to o u r

352 TABLE II Scr~war and RFLP typing t)f I,. ttlonot3'log~'ltt'.sstrains isolated during a suspected outhrcak of listcriosis Source Iiuman (n +'=

2)

Food (n = I I ) ogem fish

Scrovar

RFLP types

4

6(2) h

I/2

8(2)

ovegetarian pale

I/2

8(3)

ebcan sprouts

4

8(2)

ered cod

4

6( I), 7( I )

eice

4

6(2)

4 4

6(2) 6(2)

Environment (n = omcsh filter site maker

2)

+' "n" denotes the number of isolates. (n) h represents the numl)cr of isolates of each RFLP type from each source. R F L P type 6 also d o m i n a t e s amongst serovar 4 L. monocytogenes strains isolated in Britain.

El~idemioh~gically related strah~s T h r e e R F L P types were identified a m o n g the 17 strains of L. mwlocytogenes isohtted during a suspected o u t b r e a k of listeriosis ('Fable li). T h e h u m a n isolates were of the same serovar and R F L P type, and this combination was also found in red cod, ice and the environmental isolates. Serovar 4 strains were isolated from o ' h e r food samples although their R F L P patterns were distinct. O t h e r isolates differed in both serovar and K F L P pattern. Four pairs of h u m a n isolates (maternal-tbctal cases) were clinically related, these comprised two pairs of scrovar 1 / 2 strains ( R F L P types 2 and 4) and two pairs of scrovar 4 strains ( R F L P type 6), each pair producing identical R F L P patterns. T h e elevcn ice c r e a m isolates and four environmental isolates of L. monocytogenes obtained from a survey of an ice c r e a m factory were typed in an a t t e m p t to d e t e r m i n e potential sources of contamination. T h e isolates were g r o u p e d into three R F L P types. Serovar 4, R F L P type 6 and serovar 1/2, type 9 were isolated from the ice c r e a m and from the factory proccssing environmcnt. An additional R F L P pattern, type I I belonging to scrovar 1/2, was found in the e n v i r o n m e n t but was not detected a m o n g the ice c r e a m strains.

Discussion

T h e use of D N A p r o b c s to analysc R F L P s of Australian isolatcs of L. monocytogenes provided an increased discrimination when typing strains in epidemiological studies. Analysis of these strains indicated that the R F L P types did

353 not directly correlate with the serovars of the isolates although certain types appeared to dominate among isolates from some sources and were most commonly associated with particular serovars. The diversity of RFLP types among serovar 3 strains was high; however, isolates of this serovar are rarely implicated in cases of human iisteriosis and only seven isolates of this serovar were analysed in this study. There was a larger diversity in RFLP types detected among serovar 1/2 strains in comparison to serovar 4, both these serovars being commonly implicated in human listeriosis cases. These results are similar to those of a survey of RFLP types of British L. monocytogenes isolates by Saunders et ai. (1989) using the same probes, where greater discrimination of strains was also possible with the serovar 1 / 2 strains. Similar results with RFLP typing have been reported by other authors (Wesley et al., 1990), and Bibb et al. (1990) reported a greater genetic diversity amongst L. monocytogenes serovar 1/2 isolates analysed by multiiocus enzyme electrophoresis (MEE), also implying that serovar 4 strains of L. monocytogenes may be genetically conserved in comparison to serovar 1/2 types. Only one RFLP type was observed to be common to clinical, food and environmental sources, with many of the types being unique to fcmd and environmental or to clinical sources only. An American RFLP study (unrelated to this study) has also revealed that a particular strain of L. monocytogenes with the potential to cause outbreaks can often be quite widespread (Wesley and Ashton, 1991). Another interesting observation was the similarity of Australian RFLP types to the British types and the dominance of the same RFLP types in two geographically distant countries. This indicates the potential for wide application of the method using the currently designated types. RFLP analysis was useful in discriminating isolates of the same serovar and from the same source, into further types (e.g., red cod isolates, ice cream processing environment) thus emphasising the importance of typing more than one colony from each isolation plate for epidemiological screening purposes. Food has been implicated as a source of infection in human listeriosis. A wide variety ~f f~ods are naturally contaminated and provide a suitable reservoir, particularly as the organism is able to grow at 4°C and is relatively resistant to nitrites and salt, commonly used substances in the preservation of foods (McLauchlin et al., 1986). The human cases included here originated from an investigation of a cluster of listeriosis cases which occurred over a defined period. A common food source was sought together with other common reservoirs of infection. The occurrence of an outbreak was not confirmed and the cluster of cases resemble a phenomenon reported by others (Rocourt et al., 1989). However, RFLP analysis was a useful epidemiological tool as it allowed the exclusion of some of the suspect food a n d / o r environmental vehicles and more importantly it showed that the detection of the same serovar from a food was not sufficient to conclude it was the source. Comparison of serovar of L. monocytogenes alone often does not distinguish strains, particularly those with epidemiological associations. Further discrimination has been obtained with phage typing but the results have depended on the number and serovars of the strains typed, and the bacteriophage strains used for typing. The proportion of phage-typable ~trains of the serovars 1/2 and 4 has ranged from

354

61% (Audurier and Martin, 1989) to 92% (Loessner and Busse, 1990), indicating that until an international phage typing scheme is available, it is difficult to generalise about its usefulness. Nevertheless, Kerr et al. (1988) reported two human cases of listeriosis that were epidemiologically linked with foodstuffs by showing that both patients and food isolates were of the same serovar and phage type. in contrast to phage typing, all strains are potentially typable by DNA restriction-enzyme-fragment analysis (McLauchlin et al., 1988; Szabo and Desmarchelier, 1990) or multilocus enzyme electrophoresis analysis (Bibb et al., 1990). The disadvantages of phage typing include: varying degrees of strain susceptibility, the practical problems in the establishment/accessibility and maintenance of a bacteriophage set, and the difficulties in establishing a standard protocol. MEE has proven to be potentially useful in strain differentiation but requires analysis by a large number of enzymes. The development of rapid DNA extraction techniques, the use of DNA probes, and the development of computer generated molecular weight analyses programs, have increased the ease of discrimination possible in RFLP analysis and in the comparison of RFLP patterns. It is not proposed that RFLP analysis should substitute for serotyping but that more than one subtyping method might be employed for the optimal discrimination of strains in investigations such as suspected listeriosis outbreaks and studies of food processing environments (i.e., hazard analysis critical control point (HACCP) studies). This conclusion confirms that of similar studies where the one typing scheme alone has not been completely conclusive (Nocera et al., 1990; Szabo and Desmarchelier, 1990). Preliminary screening by a simple procedure such as serotyping, followed by other more complex methods such as RFLP analysis provide useful epidemiological tools for L. monocytogenes studies. The evaluation of this typing scheme should be pursued further, particularly when applied to epidemiological investigations aimed at targeting contamination sources.

Acknowledgements The authors wish to acknowledge Dr N.A. Saunders of the CPH laboratory, London, UK, for the supply of the probes used in this study. Thanks also to Anne Ridley of the same laboratory for a comparative analysis of our representative types with the types isolated by the CPH laboratory. The authors also wish to acknowledge the following for the donation of the Australian isolates used in this study: Mrs G. Arnold, Division of Analytical Laboratories, Sydney; Mr. T. Colby, Commonwealth Pathology, Toowoomba; Dr C. Fernandes, Royal North Shore Hospital, Sydney; Dr J. Faogali, Royal Brisbane Hospital, Queensland; Dr F. Grau, CSIRO Meat Research, Cannon Hill; Mr T. Mollee, Mater Hospital, Queensland; Mr P. Sutherland, New South Wales Dairy Corporation; Mr Y. Varabioff, Queensland Department of Primary Industries; and Dr G. De Cean, Health Department, Northern New South Wales Region.

355

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