Acta Microbiologica et Immunologica Hungarica, 61 (1), pp. 71–78 (2014) DOI: 10.1556/AMicr.2014.1111 First published online 27 December 2013
SEROGROUPING AND PULSED-FIELD GEL ELECTROPHORESIS ANALYSIS OF LISTERIA MONOCYTOGENES ISOLATES FROM CASES OF HUMAN INFECTION IN HUNGARY 2004–2012 MOLECULAR TYPING OF LISTERIA MONOCYTOGENES IN HUNGARY JUDIT PÁSZTI1, JÓZSEFNÉ KIRÁLY1 and MIKLÓS FÜZI2* 1
National Center for Epidemiology, Department of Phage Typing and Molecular Epidemiology, Budapest, Hungary 2 Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
(Received: 15 December 2013; accepted: 20 December 2013)
Eighty isolates of Listeria monocytogenes cultured from human infections in Hungary between 2004 and 2012 were serotyped by the PCR technique of Doumith et al. [9] and characterised by pulsed-field gel electrophoresis (PFGE). Most of the isolates belonged to two serogroups: 53 isolates (66.3%) to serovar group 4b,4d,4e and 21 isolates (25.8%) to serogroup 1/2a,3a. Although many pulsotypes were identified a particular pulsotype proved highly excelling comprising of 31 isolates after digestion by both ApaI and AscI restriction enzymes. All strains from this pulsotype belonged to serovar group 4b,4d,4e. Interestingly 24% of isolates from invasive samples (cerebrospinal fluid, blood) belonged to two distinct pulsotypes in the less common serovar group 1/2a,3a. Several small clusters of cases caused by isolates with identical pulsotypes were identified. Keywords: Listeria monocytogenes, serotyping, pulsotype, invasive, cluster
Introduction Listeria monocytogenes is a Gram-positive bacterial pathogen capable of causing severe invasive disease, primarily in immunocompromised patients, in the elderly, infants and pregnant women, featuring generalised infection, meningitis and miscarriage [1]. Moreover, the ingestion of large quantities of the pathogen * Corresponding author; E-mail: [email protected]
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may elicit gastroenteritis in immunocompetent individuals [2]. In Hungary listeriosis has become uncommon [3], however, infections are often associated with severe symptoms and pose a serious health hazard to the patient. Though in the industrialised countries the majority of human cases of listeriosis cannot be linked to a common source [4, 5], the often extensive geographical dissemination of cases of food-borne listeriosis [6] make the epidemiological investigation difficult. Consequently only surveillance-based molecular typing of L. monocytogenes isolates may allow for a proper evaluation of the epidemiological situation [7, 8]. Our study is a retrospective analysis of 80 L. monocytogenes isolates from cases of human infection in Hungary referred to the national reference laboratory.
Materials and Methods Strains were isolated by diagnostic microbiology laboratories across Hungary between January 2004 and December 2012 from human infections and sent to the reference laboratory at the National Center for Epidemiology. The dates and places of isolation of individual strains are shown in Table I. Samples yielding L. monocytogenes are also shown in Table I. Most of the patients suffered from invasive infections: 35 strains were isolated from CSF, 29 from blood, 3 from ascites and 1-1 from brain abscess and peritoneal dialysis fluid, respectively. The rest of the strains were cultured from non-invasive samples (Table I). All strains were serotyped by the PCR technique described by Doumith et al. [9] and characterised by PFGE using separately ApaI and AscI restriction enzymes according to the standardised Pulse-Net protocol [10, 11]. Data analysis was performed with Fingerprinting II (BioRad). Pulsotypes were defined as differing from other types by at least two bands for each individual enzyme and designated based on National Macrorestriction Profile Database (LMAPA/ASC-H). All chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, United States). Results Serotyping and PFGE analysis are currently used by many microbiology laboratories worldwide for serogrouping and subtyping L. monocytogenes. The first method assigns L. monocytogenes isolates belonging to the twelve serovars to
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five diverse serovar groups. Then the high discriminatory power of PFGE allows for further characterisation of the isolates. All results are shown and summarised in Tables I–V and Figure 1. The 80 isolates were assigned to four serovar groups by multiplex PCR. 66.0% of the strains belonged to group 4b,4d,4e and 26.0% to group 1/2a,3a (Table II). The pulsotype distribution of the isolates obtained with restriction endonuclease ApaI and AscI are shown in detail in Table I. There was agreement Table I Isolation data and typing results of individual L. monocytogenes strains Sample
Year of isolation
ear blood CSF blood blood ND blood CSF blood ear blood CSF CSF CSF blood CSF CSF CSF CSF CSF blood ND CSF ear stomach lochia blood ascites CSF blood blood CSF CSF blood CSF
2004 2005 2005 2005 2006 2006 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009
Place of isolation ND Zala Zala Budapest Borsod Borsod Budapest Tolna Borsod Szolnok Budapest Békés Szolnok Szolnok Gyõr Békés Budapest Budapest ND Budapest Pest ND Veszprém Komárom Komárom Komárom Békés Veszprém Tolna Vas Gyõr Vas Borsod Csongrád Borsod
Serovar groups
Pulsotype Apal
4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e
LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-007 LMAPA-H-002 LMAPA-H-020 LMAPA-H-001 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-025 LMAPA-H-004 LMAPA-H-022 LMAPA-H-012 LMAPA-H-001 LMAPA-H-005 LMAPA-H-025 LMAPA-H-025 LMAPA-H-003 LMAPA-H-024 LMAPA-H-001 LMAPA-H-022 LMAPA-H-022 LMAPA-H-022 LMAPA-H-020 LMAPA-H-010 LMAPA-H-022 LMAPA-H-003 LMAPA-H-003 LMAPA-H-025 LMAPA-H-020 LMAPA-H-021 LMAPA-H-020
Pulsotype Ascl LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-006 LMASC-H-005 LMASC-H-011 LMASC-H-002 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-012 LMASC-H-004 LMASC-H-026 LMASC-H-012 LMASC-H-007 LMASC-H-004 LMASC-H-012 LMASC-H-012 LMASC-H-022 LMASC-H-009 LMASC-H-018 LMASC-H-025 LMASC-H-025 LMASC-H-025 LMASC-H-011 LMASC-H-003 LMASC-H-025 LMASC-H-001 LMASC-H-022 LMASC-H-012 LMASC-H-011 LMASC-H-023 LMASC-H-011
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Table I (cont.) Sample blood dialysis fluid CSF CSF CSF ND CSF blood blood CSF blood blood blood blood blood CSF blood CSF blood CSF blood blood CSF blood blood CSF CSF blood ascites brain abscessus CSF ascites CSF blood CSF CSF blood urine CSF CSF CSF skin blood CSF blood stomach blood
Year of isolation
Place of isolation
Serovar groups
Pulsotype Apal
Pulsotype Ascl
2009 2009 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011
Csongrád Budapest Budapest Békés Gyõr ND Békés Békés Budapest Nógrád Budapest Budapest Budapest Fejér Heves Gyõr ND Budapest Gyõr Budapest Szabolcs Vas Fejér Szolnok Budapest Budapest Budapest Komárom Tolna Budapest
4b,4d,4e 4b,4d,4e 1/2b,3b,7 4b,4d,4e 4b,4d,4e 1/2c,3c 4b,4d,4e 1/2b,3b,7 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a
LMAPA-H-021 LMAPA-H-022 LMAPA-H-008 LMAPA-H-020 LMAPA-H-020 LMAPA-H-017 LMAPA-H-020 LMAPA-H-026 LMAPA-H-003 LMAPA-H-020 LMAPA-H-020 LMAPA-H-001 LMAPA-H-020 LMAPA-H-028 LMAPA-H-001 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-025 LMAPA-H-022 LMAPA-H-031 LMAPA-H-027 LMAPA-H-003 LMAPA-H-022 LMAPA-H-020 LMAPA-H-020 LMAPA-H-034 LMAPA-H-001
LMASC-H-023 LMASC-H-020 LMASC-H-027 LMASC-H-011 LMASC-H-011 LMASC-H-021 LMASC-H-011 LMASC-H-024 LMASC-H-001 LMASC-H-011 LMASC-H-011 LMASC-H-007 LMASC-H-011 LMASC-H-010 LMASC-H-007 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-012 LMASC-H-014 LMASC-H-016 LMASC-H-012 LMASC-H-015 LMASC-H-013 LMASC-H-011 LMASC-H-011 LMASC-H-020 LMASC-H-018
2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2012 2012
ND Budapest Budapest Békés Komárom Borsod Tolna ND Szolnok Pest ND Borsod Budapest Veszprém Borsod Szabolcs Budapest
1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2b,3b,7 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e
LMAPA-H-033 LMAPA-H-020 LMAPA-H-035 LMAPA-H-020 LMAPA-H-020 LMAPA-H-033 LMAPA-H-036 LMAPA-H-032 LMAPA-H-035 LMAPA-H-020 LMAPA-H-022 LMAPA-H-020 LMAPA-H-036 LMAPA-H-020 LMAPA-H-003 LMAPA-H-025 LMAPA-H-020
LMASC-H-014 LMASC-H-011 LMASC-H-019 LMASC-H-011 LMASC-H-011 LMASC-H-007 LMASC-H-010 LMASC-H-017 LMASC-H-028 LMASC-H-011 LMASC-H-025 LMASC-H-011 LMASC-H-010 LMASC-H-011 LMASC-H-001 LMASC-H-028 LMASC-H-011
PFGE was performed with two distinct restriction endonucleases: ApaI and AscI; ND: no data; CSF: cerebrospinal fluid
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between results obtained with the respective enzymes. The frequency of individual pulsotypes is summarised in Table III. As shown there were five pulsotypes excelling in frequency (pulsotypes LMAPA-H-020, LMAPA-H-022, LMAPA-H-025 and LMAPA-H-001, LMAPA-H-003). All isolates with pulsotypes LMAPA-H-020, LMAPA-H-022 and LMAPA-H-025 belonged to serovar group 4b,4d,4e and all isolates with pulsotype LMAPA-H-001 and LMAPA-H-003 belonged to serogroup 1/2a,3a (Table I). LMAPA-H-020/ LMASC-H-011 (31) and LMAPA-H-025/LMASC-H-012 proved the most frequent pulsotypes with both enzymes. Table II Distribution of L. monocytogenes isolates by serotype Serovar groups
Number
1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e
21 (26.0%) 4 (5.0%) 2 (3.0%) 53 (66.0%)
All
80 (100%) Table III
Number of L. monocytogenes isolates assigned to individual pulsotypes (ApaI and AscI) Pulsotype ApaI/AscI
LMASC-H-007
LMAPA-H-001 LMAPA-H-003 LMAPA-H-020 LMAPA-H-022 LMAPA-H-025 LMAPA-H-032 Other
3
Total
4
LMASC-H-011
LMASC-H-012 LMASC-H-025
Other Total 3 6
5
4 1 1 18
6 6 31 9 6 1 21
5
33
80
31 5 1
2 31
7
The number of strains from each serogroup and from the most common pulsotypes causing invasive infections are displayed in Table IV and Table V, respectively. Pulsotype LMAPA-H-22 proved somewhat less pathogenic than other major pulsotypes (Table V).
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Table IV Distribution of L. monocytogenes strains isolated from invasive samples by serotype Serovar groups
Sample type
1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e
Blood
CSF
All
9 4
10 1
20
29
23 5 2 59
CSF: cerebrospinal fluid Table V Number of L. monocytogenes strains from the most common pulsotypes isolated from invasive samples Sample type
Pulsotype (Apal) Blood
CSF
All
Invasive%
LMAPA-H-20 LMAPA-H-22 LMAPA-H-01 LMAPA-H-03 LMAPA-H-25
12 2 2 6 1
15 3 4* – 4
31 10 6 6 6
87 50 100 100 83
Total
23
26
59
83
* One of the strains was isolated from a brain abscess; CSF: cerebrospinal fluid
Discussion We have for the first time serotyped and characterised by PFGE a large number of L. monocytogenes strains isolated from human samples in Hungary. Some L. monocytogenes isolates of human origin were serotyped in 2006 [12]. Then 1/2a (45.1%) and 4/b (27.0%) proved the most prevalent serotypes [12]. In 2008 a connatal case of listeriosis was reported from Hungary [13]. Listerisosis has become uncommon in Hungary: the number of reported cases remained between 9 and 20 annually in the past decade [3]. However, as our patients also attest the cases of listeriosis in Hungary are mostly severe generalised infections. The results of serotyping show that in Hungary the dominant serovar group of L. monocytogenes causing infections in the last couple of years has been: 4b,4d,4e. The PFGE characterisation of the isolates revealed that a particular pulsotype – LMAPA-H-020/LMASC-H-011 – is most common in human infections. A considerable proportion (87%) of the pulsotype LMAPA-H-020/ LMASC-H-011 strains (Table V) were isolated from invasive infections. 24% of
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isolates from invasive samples (cerebrospinal fluid, blood) belonged to two distinct pulsotypes (LMAPA-H-01 and LMAPA-H-03) in the less common serovar group 1/2a,3a (Table I, Table V). Though only two family outbreaks of listeriosis were identified in Hungary in the study period, the temporal and geographical distribution of the isolates suggests that a few cases might have been related. There were multiple isolations of L. monocytogenes with identical pulsotypes: in 2005 (Zala county), 2009 (Komárom county and Békés county) and 2011 (Komárom county) (Table I, Figure 1). Our results demonstrate that the dissemination of L. monocytogenes in Hungary is partly clonal and the virulence of individual clones might be diverse. In addition, the multiple occurrence of cases of listeriosis caused by strains with identical pulsotypes in close temporal and geographical proximity necessitates a continuous thorough epidemiological investigation of the situation.
2005 2008
2009
2011 2012
2009
2009
2010 2009 2011
2010
2012
2007
2010
2012 2007
2012
2010
2010
2007
2010
2008
2008
2009 2005
2011
2009
4b,4d,4e LMAPA-H-020/ LMASC-H-011
1/ 2a LMAPA-H-003/ LMASC-H-001 LMAPA-H-001/ LMASC-H-007
LMAPA-H-025/ LMASC-H-012 LMAPA-H-022/ LMASC-H-025
Figure 1. Temporal and geographical distribution of L. monocytogenes pulsotypes in Hungary
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References 1. Drevets, D.A., Bronze, M.S.: Listeria monocytogenes: Epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 53, 151–165 (2008). 2. Ooi, S.T., Lorber, B.: Gastroenteritis due to Listeria monocytogenes. Clin Infect Dis 40, 1327–1332 (2005). 3. www.oek.hu 4. La Scola, B., Fournier, P.E., Musso, D., Tissot-Dupont, H.: Pseudo-outbreak of listeriosis elucidated by pulsed-field gel electrophoresis. Eur J Clin Microbiol Infect Dis 16, 756–760 (1996). 5. Sauders, B.D., Fortes, E.D., Morse, D.L., Dumas, N., Kiehlbauch, J.A., Schukken, Y., Hibbs, J.R., Wiedmann, M.: Molecular subtyping to detect human listeriosis clusters. Emerg Infect Dis 9, 672–680 (2003). 6. Sauders, B.D., Schukken, Y., Kornstein, L., Reddy, V., Bannerman, T., Salehi, E., Dumas, N., Anderson, B.J., Massey, J.P., Wiedmann, M.: Molecular epidemiology and cluster analysis of human listeriosis cases in three U.S. states. J Food Prot 69, 1680–1689 (2006). 7. Fugett, E.B., Schoonmaker-Bopp, D., Dumas, N.B., Corby, J., Wiedmann, M.: Pulsed-field gel electrophoresis (PFGE) analysis of temporally matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms, and urban and natural environments reveals source-associated as well as widely distributed PFGE types. J Clin Microbiol 45, 865–873 (2007). 8. Cartwright, E.J., Jackson, K.A., Johnson, S.D., Graves, L.M., Silk, B.J., Mahon, B.E.: Listeriosis outbreaks and associated food vehicles, United States, 1998–2008. Emerg Infect Dis 19, 1–9 (2013). 9. Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C., Martin, P.: Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol 42, 3819–3822 (2004). 10. Graves, L.M., Swaminathan, B.: PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int J Food Microbiol 65, 55–62 (2001). 11. http://www.pulsenetinternational.org/assets/PulseNet/uploads/pfge/PNL04_ListeriaPFGE Protocol.pdf 12. Kiss, R., Tirczka, T., Szita, G., Bernáth, S., Csikó Gy.: Listeria monocytogenes food monitoring data and incidence of human listeriosis in Hungary, 2004. International Journal of Food Microbiology 112, 71–74 (2006). 13. Kristóf, K., Barcs, I., Cziniel, M., Ghidán, A., Nagy, K.: Connatal listeriosis – A case report and the possibilities of microbiological diagnosis. Acta Microbiol Immunol Hung 55, 63–72 (2008).
Acta Microbiologica et Immunologica Hungarica 61, 2014
SEROGROUPING AND PULSED-FIELD GEL ELECTROPHORESIS ANALYSIS OF LISTERIA MONOCYTOGENES ISOLATES FROM CASES OF HUMAN INFECTION IN HUNGARY 2004–2012 MOLECULAR TYPING OF LISTERIA MONOCYTOGENES IN HUNGARY JUDIT PÁSZTI1, JÓZSEFNÉ KIRÁLY1 and MIKLÓS FÜZI2* 1
National Center for Epidemiology, Department of Phage Typing and Molecular Epidemiology, Budapest, Hungary 2 Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
(Received: 15 December 2013; accepted: 20 December 2013)
Eighty isolates of Listeria monocytogenes cultured from human infections in Hungary between 2004 and 2012 were serotyped by the PCR technique of Doumith et al. [9] and characterised by pulsed-field gel electrophoresis (PFGE). Most of the isolates belonged to two serogroups: 53 isolates (66.3%) to serovar group 4b,4d,4e and 21 isolates (25.8%) to serogroup 1/2a,3a. Although many pulsotypes were identified a particular pulsotype proved highly excelling comprising of 31 isolates after digestion by both ApaI and AscI restriction enzymes. All strains from this pulsotype belonged to serovar group 4b,4d,4e. Interestingly 24% of isolates from invasive samples (cerebrospinal fluid, blood) belonged to two distinct pulsotypes in the less common serovar group 1/2a,3a. Several small clusters of cases caused by isolates with identical pulsotypes were identified. Keywords: Listeria monocytogenes, serotyping, pulsotype, invasive, cluster
Introduction Listeria monocytogenes is a Gram-positive bacterial pathogen capable of causing severe invasive disease, primarily in immunocompromised patients, in the elderly, infants and pregnant women, featuring generalised infection, meningitis and miscarriage [1]. Moreover, the ingestion of large quantities of the pathogen * Corresponding author; E-mail: [email protected]
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may elicit gastroenteritis in immunocompetent individuals [2]. In Hungary listeriosis has become uncommon [3], however, infections are often associated with severe symptoms and pose a serious health hazard to the patient. Though in the industrialised countries the majority of human cases of listeriosis cannot be linked to a common source [4, 5], the often extensive geographical dissemination of cases of food-borne listeriosis [6] make the epidemiological investigation difficult. Consequently only surveillance-based molecular typing of L. monocytogenes isolates may allow for a proper evaluation of the epidemiological situation [7, 8]. Our study is a retrospective analysis of 80 L. monocytogenes isolates from cases of human infection in Hungary referred to the national reference laboratory.
Materials and Methods Strains were isolated by diagnostic microbiology laboratories across Hungary between January 2004 and December 2012 from human infections and sent to the reference laboratory at the National Center for Epidemiology. The dates and places of isolation of individual strains are shown in Table I. Samples yielding L. monocytogenes are also shown in Table I. Most of the patients suffered from invasive infections: 35 strains were isolated from CSF, 29 from blood, 3 from ascites and 1-1 from brain abscess and peritoneal dialysis fluid, respectively. The rest of the strains were cultured from non-invasive samples (Table I). All strains were serotyped by the PCR technique described by Doumith et al. [9] and characterised by PFGE using separately ApaI and AscI restriction enzymes according to the standardised Pulse-Net protocol [10, 11]. Data analysis was performed with Fingerprinting II (BioRad). Pulsotypes were defined as differing from other types by at least two bands for each individual enzyme and designated based on National Macrorestriction Profile Database (LMAPA/ASC-H). All chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, United States). Results Serotyping and PFGE analysis are currently used by many microbiology laboratories worldwide for serogrouping and subtyping L. monocytogenes. The first method assigns L. monocytogenes isolates belonging to the twelve serovars to
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five diverse serovar groups. Then the high discriminatory power of PFGE allows for further characterisation of the isolates. All results are shown and summarised in Tables I–V and Figure 1. The 80 isolates were assigned to four serovar groups by multiplex PCR. 66.0% of the strains belonged to group 4b,4d,4e and 26.0% to group 1/2a,3a (Table II). The pulsotype distribution of the isolates obtained with restriction endonuclease ApaI and AscI are shown in detail in Table I. There was agreement Table I Isolation data and typing results of individual L. monocytogenes strains Sample
Year of isolation
ear blood CSF blood blood ND blood CSF blood ear blood CSF CSF CSF blood CSF CSF CSF CSF CSF blood ND CSF ear stomach lochia blood ascites CSF blood blood CSF CSF blood CSF
2004 2005 2005 2005 2006 2006 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009
Place of isolation ND Zala Zala Budapest Borsod Borsod Budapest Tolna Borsod Szolnok Budapest Békés Szolnok Szolnok Gyõr Békés Budapest Budapest ND Budapest Pest ND Veszprém Komárom Komárom Komárom Békés Veszprém Tolna Vas Gyõr Vas Borsod Csongrád Borsod
Serovar groups
Pulsotype Apal
4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 1/2a,3a 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e
LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-007 LMAPA-H-002 LMAPA-H-020 LMAPA-H-001 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-025 LMAPA-H-004 LMAPA-H-022 LMAPA-H-012 LMAPA-H-001 LMAPA-H-005 LMAPA-H-025 LMAPA-H-025 LMAPA-H-003 LMAPA-H-024 LMAPA-H-001 LMAPA-H-022 LMAPA-H-022 LMAPA-H-022 LMAPA-H-020 LMAPA-H-010 LMAPA-H-022 LMAPA-H-003 LMAPA-H-003 LMAPA-H-025 LMAPA-H-020 LMAPA-H-021 LMAPA-H-020
Pulsotype Ascl LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-006 LMASC-H-005 LMASC-H-011 LMASC-H-002 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-012 LMASC-H-004 LMASC-H-026 LMASC-H-012 LMASC-H-007 LMASC-H-004 LMASC-H-012 LMASC-H-012 LMASC-H-022 LMASC-H-009 LMASC-H-018 LMASC-H-025 LMASC-H-025 LMASC-H-025 LMASC-H-011 LMASC-H-003 LMASC-H-025 LMASC-H-001 LMASC-H-022 LMASC-H-012 LMASC-H-011 LMASC-H-023 LMASC-H-011
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Table I (cont.) Sample blood dialysis fluid CSF CSF CSF ND CSF blood blood CSF blood blood blood blood blood CSF blood CSF blood CSF blood blood CSF blood blood CSF CSF blood ascites brain abscessus CSF ascites CSF blood CSF CSF blood urine CSF CSF CSF skin blood CSF blood stomach blood
Year of isolation
Place of isolation
Serovar groups
Pulsotype Apal
Pulsotype Ascl
2009 2009 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011
Csongrád Budapest Budapest Békés Gyõr ND Békés Békés Budapest Nógrád Budapest Budapest Budapest Fejér Heves Gyõr ND Budapest Gyõr Budapest Szabolcs Vas Fejér Szolnok Budapest Budapest Budapest Komárom Tolna Budapest
4b,4d,4e 4b,4d,4e 1/2b,3b,7 4b,4d,4e 4b,4d,4e 1/2c,3c 4b,4d,4e 1/2b,3b,7 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a
LMAPA-H-021 LMAPA-H-022 LMAPA-H-008 LMAPA-H-020 LMAPA-H-020 LMAPA-H-017 LMAPA-H-020 LMAPA-H-026 LMAPA-H-003 LMAPA-H-020 LMAPA-H-020 LMAPA-H-001 LMAPA-H-020 LMAPA-H-028 LMAPA-H-001 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-020 LMAPA-H-025 LMAPA-H-022 LMAPA-H-031 LMAPA-H-027 LMAPA-H-003 LMAPA-H-022 LMAPA-H-020 LMAPA-H-020 LMAPA-H-034 LMAPA-H-001
LMASC-H-023 LMASC-H-020 LMASC-H-027 LMASC-H-011 LMASC-H-011 LMASC-H-021 LMASC-H-011 LMASC-H-024 LMASC-H-001 LMASC-H-011 LMASC-H-011 LMASC-H-007 LMASC-H-011 LMASC-H-010 LMASC-H-007 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-011 LMASC-H-012 LMASC-H-014 LMASC-H-016 LMASC-H-012 LMASC-H-015 LMASC-H-013 LMASC-H-011 LMASC-H-011 LMASC-H-020 LMASC-H-018
2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2012 2012
ND Budapest Budapest Békés Komárom Borsod Tolna ND Szolnok Pest ND Borsod Budapest Veszprém Borsod Szabolcs Budapest
1/2a,3a 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e 4b,4d,4e 4b,4d,4e 4b,4d,4e 1/2b,3b,7 4b,4d,4e 1/2a,3a 4b,4d,4e 4b,4d,4e
LMAPA-H-033 LMAPA-H-020 LMAPA-H-035 LMAPA-H-020 LMAPA-H-020 LMAPA-H-033 LMAPA-H-036 LMAPA-H-032 LMAPA-H-035 LMAPA-H-020 LMAPA-H-022 LMAPA-H-020 LMAPA-H-036 LMAPA-H-020 LMAPA-H-003 LMAPA-H-025 LMAPA-H-020
LMASC-H-014 LMASC-H-011 LMASC-H-019 LMASC-H-011 LMASC-H-011 LMASC-H-007 LMASC-H-010 LMASC-H-017 LMASC-H-028 LMASC-H-011 LMASC-H-025 LMASC-H-011 LMASC-H-010 LMASC-H-011 LMASC-H-001 LMASC-H-028 LMASC-H-011
PFGE was performed with two distinct restriction endonucleases: ApaI and AscI; ND: no data; CSF: cerebrospinal fluid
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between results obtained with the respective enzymes. The frequency of individual pulsotypes is summarised in Table III. As shown there were five pulsotypes excelling in frequency (pulsotypes LMAPA-H-020, LMAPA-H-022, LMAPA-H-025 and LMAPA-H-001, LMAPA-H-003). All isolates with pulsotypes LMAPA-H-020, LMAPA-H-022 and LMAPA-H-025 belonged to serovar group 4b,4d,4e and all isolates with pulsotype LMAPA-H-001 and LMAPA-H-003 belonged to serogroup 1/2a,3a (Table I). LMAPA-H-020/ LMASC-H-011 (31) and LMAPA-H-025/LMASC-H-012 proved the most frequent pulsotypes with both enzymes. Table II Distribution of L. monocytogenes isolates by serotype Serovar groups
Number
1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e
21 (26.0%) 4 (5.0%) 2 (3.0%) 53 (66.0%)
All
80 (100%) Table III
Number of L. monocytogenes isolates assigned to individual pulsotypes (ApaI and AscI) Pulsotype ApaI/AscI
LMASC-H-007
LMAPA-H-001 LMAPA-H-003 LMAPA-H-020 LMAPA-H-022 LMAPA-H-025 LMAPA-H-032 Other
3
Total
4
LMASC-H-011
LMASC-H-012 LMASC-H-025
Other Total 3 6
5
4 1 1 18
6 6 31 9 6 1 21
5
33
80
31 5 1
2 31
7
The number of strains from each serogroup and from the most common pulsotypes causing invasive infections are displayed in Table IV and Table V, respectively. Pulsotype LMAPA-H-22 proved somewhat less pathogenic than other major pulsotypes (Table V).
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Table IV Distribution of L. monocytogenes strains isolated from invasive samples by serotype Serovar groups
Sample type
1/2a,3a 1/2b,3b,7 1/2c,3c 4b,4d,4e
Blood
CSF
All
9 4
10 1
20
29
23 5 2 59
CSF: cerebrospinal fluid Table V Number of L. monocytogenes strains from the most common pulsotypes isolated from invasive samples Sample type
Pulsotype (Apal) Blood
CSF
All
Invasive%
LMAPA-H-20 LMAPA-H-22 LMAPA-H-01 LMAPA-H-03 LMAPA-H-25
12 2 2 6 1
15 3 4* – 4
31 10 6 6 6
87 50 100 100 83
Total
23
26
59
83
* One of the strains was isolated from a brain abscess; CSF: cerebrospinal fluid
Discussion We have for the first time serotyped and characterised by PFGE a large number of L. monocytogenes strains isolated from human samples in Hungary. Some L. monocytogenes isolates of human origin were serotyped in 2006 [12]. Then 1/2a (45.1%) and 4/b (27.0%) proved the most prevalent serotypes [12]. In 2008 a connatal case of listeriosis was reported from Hungary [13]. Listerisosis has become uncommon in Hungary: the number of reported cases remained between 9 and 20 annually in the past decade [3]. However, as our patients also attest the cases of listeriosis in Hungary are mostly severe generalised infections. The results of serotyping show that in Hungary the dominant serovar group of L. monocytogenes causing infections in the last couple of years has been: 4b,4d,4e. The PFGE characterisation of the isolates revealed that a particular pulsotype – LMAPA-H-020/LMASC-H-011 – is most common in human infections. A considerable proportion (87%) of the pulsotype LMAPA-H-020/ LMASC-H-011 strains (Table V) were isolated from invasive infections. 24% of
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isolates from invasive samples (cerebrospinal fluid, blood) belonged to two distinct pulsotypes (LMAPA-H-01 and LMAPA-H-03) in the less common serovar group 1/2a,3a (Table I, Table V). Though only two family outbreaks of listeriosis were identified in Hungary in the study period, the temporal and geographical distribution of the isolates suggests that a few cases might have been related. There were multiple isolations of L. monocytogenes with identical pulsotypes: in 2005 (Zala county), 2009 (Komárom county and Békés county) and 2011 (Komárom county) (Table I, Figure 1). Our results demonstrate that the dissemination of L. monocytogenes in Hungary is partly clonal and the virulence of individual clones might be diverse. In addition, the multiple occurrence of cases of listeriosis caused by strains with identical pulsotypes in close temporal and geographical proximity necessitates a continuous thorough epidemiological investigation of the situation.
2005 2008
2009
2011 2012
2009
2009
2010 2009 2011
2010
2012
2007
2010
2012 2007
2012
2010
2010
2007
2010
2008
2008
2009 2005
2011
2009
4b,4d,4e LMAPA-H-020/ LMASC-H-011
1/ 2a LMAPA-H-003/ LMASC-H-001 LMAPA-H-001/ LMASC-H-007
LMAPA-H-025/ LMASC-H-012 LMAPA-H-022/ LMASC-H-025
Figure 1. Temporal and geographical distribution of L. monocytogenes pulsotypes in Hungary
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