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Use of PFGE to characterize clonal relationships among Belgian clinical isolates of Listeria monocytogenes

Listeria Reference Centre, Section of Bacteriology, Institute of Public Health, J. Wytsmanstraat 16, B-1050 Brussels, Belgium

Correspondence Marc Yde Marc.Yde{at}iph.fgov.be

Received June 20, 2003
Accepted January 19, 2004

The Belgian Listeria Reference Centre receives between 30 and 50 human clinical strains of Listeria monocytogenes per year. In general, epidemiological data are absent or incomplete, preventing recognition of episodes of listeriosis. However, data on a clonal relationship between strains can indirectly give an idea of the occurrence of episodes. Human isolates of L. monocytogenes from 2001 were serotyped, their arsenic-cadmium resistance profiles were determined, and they were pulsotyped with the application of pulsed-field gel electrophoresis using AscI and ApaI restriction endonucleases. On five occasions, two or more strains presented the same serovar, metal-resistance profile and pulsovar, suggesting a clonal relationship. This is the first report to identify accurately potential listeriosis episodes occurring in Belgium.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Human listeriosis can occur either as a sporadic disease (probably most often of foodborne origin) or as an outbreak. Most cases of listeriosis are considered to be single cases. The incubation period and infective dose have not been firmly established, although they may be inversely related. Reported incubation times vary from a few days to 2–3 months. Outbreaks may therefore easily be missed. The annual rate varies from 2 to 15 cases per million population (Rocourt & Bille, 1997). However, the high-risk population is increasing (elderly, immunocompromised patients, transplant patients, HIV-positive patients), particularly in the Western hemisphere. Likewise, the potential sources of listeriosis from contaminated food have also increased because of major changes in food production, preservation and consumption. The identification and investigation of outbreaks of human listeriosis require knowledge of the microbiological characteristics of clinical and environmental isolates. Various methods have been developed for such investigations: serotyping (Seeliger & Hohne, 1979), bacteriophage typing (Loesser & Busse, 1990), rDNA fingerprinting (Graves et al., 1991), restriction fragment length polymorphism analysis (Ridley, 1995), multilocus enzyme electrophoresis (Selander et al., 1986), pulsed-field gel electrophoresis (PFGE) (Brosch et al., 1991) and randomly amplified polymorphic DNA patterns (Mazurier et al., 1992).

The Belgian Listeria Reference Centre receives between 30 and 50 human clinical strains of Listeria monocytogenes per year. The aim of this study was to investigate clonal relationships between clinical strains of L. monocytogenes isolated in Belgium in 2001. All strains were subtyped by PFGE. This technique was preferred because it has been established that PFGE is a highly discriminatory and reproducible method for subtyping L. monocytogenes (Brosch et al., 1996). In addition, this technique was chosen by the US PulseNet, a national network of public health laboratories that fingerprint foodborne bacteria, such as L. monocytogenes (Swaminathan et al., 2001).

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Forty-eight strains of L. monocytogenes isolated from human cases of listeriosis in Belgium in 2001 were available for investigation. The strains were serotyped according to the reference method (Seeliger & Hohne, 1979). Strains were further subtyped on the basis of arsenic and cadmium susceptibility according to McLauchlin et al. (1997) with minor modifications: the results were read after 48 h instead of overnight incubation because of delayed growth with some strains. The protocol of Graves & Swaminathan (2001) for subtyping L. monocytogenes by macrorestriction and PFGE was used. DNA restriction fragments in plugs were separated by electrophoresis on a Gene Navigator PFGE apparatus (Pharmacia-LKB). DNA restriction fragments were sized against lambda DNA ladder (Boehringer). L. monocytogenes strain ATCC 51777 was used as the standard/reference strain. Analysis of banding patterns was performed with ImageMaster video documentation system (Amersham Pharmacia Biotech) and ImageMaster 1D software, version 4.00 (Amersham Pharmacia Biotech).

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Serotyping

Of the 48 strains of L. monocytogenes investigated, 26 belonged to serovar 4b, 18 to serovar 1/2a and four to serovar 1/2b. This partition is not unusual because most (> 95 %) human infections are caused by strains belonging to serovars 4b, 1/2a and 1/2b (Graves et al., 1999). Therefore, serotyping alone is of limited value in epidemiological investigations.

Arsenic and cadmium susceptibility

Subtyping based on arsenic and cadmium susceptibility is easy to perform and can subdivide strains grouped within the same serovar. The results for 48 clinical strains of human origin are shown in Table 1. Strains belonging to serovar 4b could be subdivided into four biotypes: resistant to arsenic and cadmium (A^RC^R), resistant to arsenic and sensitive to cadmium (A^RC^S), sensitive to arsenic and cadmium (A^SC^S) and sensitive to arsenic and resistant to cadmium (A^SC^R). It is noteworthy that no arsenic-resistant strains were detected among members of serogroup 1/2. McLauchlin et al. (1997) also reported small numbers of serogroup 1/2 clinical isolates resistant to arsenic.

Pulsotyping

Strains of L. monocytogenes were grouped on the basis of serovar and arsenic-cadmium resistance for PFGE. All isolates displayed AscI and ApaI restriction endonuclease digestion profiles (Fig. 1). AscI generated 6–12 major fragments ranging in size from approx. 25 to 675 kb, while ApaI-digested DNA generated digestion profiles with 8–21 major fragments ranging in size from 20 to 860 kb.

View larger version (97K): [in this window] [in a new window]   Fig. 1. PFGE patterns for AscI and ApaI of representative L. monocytogenes isolates for each of the five identified listeria episodes. Lanes 1–5, isolates from episodes I–V; lane 6, reference strain ATCC 51777.

PFGE data are presented in Table 1. For every serovar-metal resistance subtype, the numbers of different AscI and ApaI profiles are given and also the number of pulsotypes. Different profiles were defined by a change of at least one band (i.e. a missing band, an extra band or migration to a different position). Of the 48 clinical strains, 34 different AscI profiles and 38 different ApaI profiles were encountered, resulting in 39 different pulsovars (81 %). There were no common pulsovars across the different serotype and metal-resistance groupings. The large number of different pulsovars indicated that a great variety of genetically distinct strains cause listeriosis in Belgium. It also points indirectly to a low degree of listeriosis outbreaks: only 14 strains of L. monocytogenes were involved in likely episodes of listeriosis (three episodes involved two cases, one episode comprised three and one episode comprised five clinical cases).

The high percentage of pulsovars in this study is in line with published data for clinical cases. Table 2 includes data from nine other studies. Great variation is seen in the number of pulsovars obtained in different studies. Several factors contribute to this phenomenon, (i) not all of the clinical strains used correspond to sporadic cases; (ii) the choice of macrorestriction endonucleases; and (iii) analysis protocol. The current work was the only study that used the PulseNet method described by Graves & Swaminathan (2001).

The necessity of two restriction enzymes

One hospital sent three strains of L. monocytogenes to the Listeria Reference Centre in a single month. The strains were isolated from elderly people, living in the same region. The three strains belonged to serovar 1/2b, an infrequent serovar among human clinical isolates. In addition, these strains presented the same metal-resistance profile (A^SC^R), suggesting an outbreak. PFGE, however, differentiated the three strains. Two of the three strains presented identical AscI profiles, but gave distinctive ApaI profiles (four bands difference). On another occasion, two strains of L. monocytogenes serotype 4b (A^RC^S) isolated within the same period displayed identical ApaI profiles but were differentiated by their AscI profiles, distinct by one band. Such a small difference indicates a single genetic event and thus suggests some relatedness. However, epidemiological data did not indicate an episode.

Characteristics of listeria episodes

PFGE identified five occasions when the same L. monocytogenes biotype was isolated from more than one person in Belgium. However, a general outbreak was not likely, as the number of infected people was small. Three episodes were linked to strains of serovar 4b, and two to serovar 1/2a strains. Three metal-resistance profiles were seen: A^RC^S (episode I), A^SC^R (episode IV) and A^SC^S (the remaining episodes). On one occasion, ice cream cake was identified as the food vehicle. Fig. 1 shows the representative profiles of each episode.

Epidemiological evidence for the episodes was not always apparent. The most prominent epidemiological evidence was obtained for two patients from episode I. Two strains (serovar 4b) were isolated from elderly patients at the same hospital on the same day. There was no obvious link with the third patient of episode I. She was younger (48 years old) and lived in another province. However, the same strain was isolated at the same time. The second episode is an example of an episode with no epidemiological evidence. The strains (serovar 4b) were isolated from a cancer patient (74 years old) and a neonate, 5 months, and 50 km apart. This incident underlines the persistence and/or spread of L. monocytogenes in the community. Episode III concerned two immunocompromised women aged 57 and 62. The same strain (serovar 1/2a) was isolated in different provinces, 6 weeks apart. No common source of infection was seen in episode IV. Five elderly men were infected by the same strain (serovar 1/2a). The strains were isolated over a period of 9 months, in four different provinces. One of the patients died of listeriosis complications. With episode V, the source of food contamination was traced. Two patients, one with meningitis and the other with gastroenteritis, attended a family party. Frozen ice cream cake was served, which subsequently was shown to contain L. monocytogenes (serovar 4b). The strains isolated from the food and from the two patients were identified as the same pulsovar.

It is clear from the description of these episodes that epidemiological evidence is generally flimsy at best. Based upon serotyping, metal-resistance profiles and pulsotyping, the Listeria Reference laboratory found clonal relatedness between strains, thereby suggesting the possibility of episodes of listeriosis. PFGE demonstrated the greatest discrimination of the three assays. Information on the patient (age, gender, address, etc.) and on the isolation of the strain (time, hospital, biological fluid, etc.) alone is insufficient. More appropriate information must be gathered regarding the patients’ food habits by interviewing them. This information is not easy to obtain, especially in Belgium, where the Flemish- and French-speaking communities have their own health inspections. Furthermore, as identical strains are sometimes isolated after a relatively long interval of time, investigators may have difficulty identifying patient histories. On the other hand, the possibility of a coincidental episode must not be ignored. According to Autio et al. (2002), the recovery of indistinguishable pulsotypes may mislead the investigation towards establishing the vehicle of infection. Several food products may harbour identical strains without any form of cross-contamination or common infection source. Therefore, a suspected epidemic may actually consist of two or more independent sporadic cases. However, as long as there is a large variation of circulating pulsovars, the probability of dealing with a coincidental episode seems low.

Human listeriosis in Belgium involves a great variety of genetically different strains. However, on five occasions in 2001, indistinguishable strains were isolated from more than one patient, suggesting the existence of five different episodes of listeriosis. In four of the five episodes, the food vehicle was not determined, because of a lack of epidemiological evidence.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We are grateful to C. Hemmerijckx for technical assistance in preparing the manuscript.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES