Heat-stable antigen serotyping of Campylobacter jejuni strains isolated from hospitalized children in Athens, Greece

A hundred and twentynine Campylobacter jejuni strains isolated from hospitalized children with gastroenteritis were serotyped by the heat-stable antigen scheme (HS, Penner's method). Isolates belonged to two different periods. Group A contained strains isolated in 1987–1988 and group B contained strains which were isolated in 1998–2000. A variety of serotypes was found. Serotype HS:2 was predominant, followed by the HS:4 complex and HS:1,44. Many clinically important Guillain-Barré Syndrome associated serotypes – like HS:19 – were identified. There were no significant differences in the distribution of serotypes between the two periods. The present report provides reference data, as this is the first C. jejuni serotyping study ever made in Greece.     

Risk factors for infection with Campylobacter jejuni flaA genotypes

We aimed to explore Campylobacter genotype-specific risk factors in Australia. Isolates collected prospectively from cases recruited into a case-control study were genotyped using flaA restriction fragment-length polymorphism typing (flaA genotyping). Exposure information for cases and controls was collected by telephone interview. Risk factors were examined for major flaA genotypes using logistic and multinomial regression. Five flaA genotypes accounted for 325 of 590 (55%) cases - flaA-6b (n=129), flaA-6 (n=70), flaA-10 (n=48), flaA-2 (n=43), flaA-131 (n=35). In Australia, infections due to flaA-10 and flaA-2 were found to be significantly associated with eating non-poultry meat (beef and ham, respectively) in both case-control and inter-genotype comparisons. All major genotypes apart from flaA-10 were associated with chicken consumption in the case-control comparisons. Based on several clinical criteria, infections due to flaA-2 were more severe than those due to other genotypes. Thus genotype analysis may reveal genotype-specific niches and differences in virulence and transmission routes.     

A simultaneous outbreak of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit

We describe two concurrent outbreaks of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit (NICU). Over a 16-month period, a total of 27 infants were either colonized (N=14) or infected (N=13). There were 15 cases of S. marcescens and 11 cases of K. pneumoniae. Both micro-organisms were involved in one fatal case. Seven preterm babies developed septicaemia, two had bacteraemia, three had respiratory infections and one had purulent conjunctivitis. The S. marcescens and K. pneumoniae isolates were investigated by three molecular methods: enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), arbitrary primed PCR with M13 primer, and random amplification of polymorphic DNA. Different patterns were found in the 16 S. marcescens epidemic isolates from 16 newborn infants. The major epidemic-involved genotype was linked to the first nine cases and this was subsequently replaced by different patterns. Eight different typing profiles were also determined for the 13 K. pneumoniae isolates from 12 newborn infants. Four K. pneumoniae bacteraemic strains proved to be identical. In conclusion, the typing results revealed that two different micro-organisms (S. marcescens and K. pneumoniae) were simultaneously involved in invasive nosocomial infections in preterm newborns. Two simultaneous clusters of cases were documented. Heterogeneous genotypes among both species were also demonstrated to be present in the NICU at the same time. A focal source for both micro-organisms was not identified but cross-transmission through handling was probably an important route in this outbreak. Strict adherence to handwashing policies, cohorting, isolation of colonized and infected patients, and rigorous environmental hygiene were crucial measures in the containment of the epidemic.     

多位点序列分型和脉冲场凝胶电泳在肠炎沙门菌分子分型的比较

目的 比较多位点序列分型技术和脉冲场凝胶电泳（PFGE）技术在肠炎沙门菌菌株间分型的分辨率。方法 分别建立肠炎沙门菌的6个管家基因thrA、pure、sucA、aroC、hemD、dnaN和一个特异性的DNA标记Sdf Ⅰ的多位点序列分型技术，及以寡切点的XbaⅠ、SpeⅠ作为限制性内切酶的PFGE方法，并应用上述方法对食品中的分离株进行分型，比较两种方法的分辨率。结果 PFGE可以将50株肠炎沙门菌分为11个型。并且通过双酶系统的双重PFGE分型，还可以将PFGE型别再精细划分为亚型；MLST则揭示了在同一血清型内部，各菌株之间的管家基因碱基序列高度保守，而在沙门菌不同血清型的核苷酸序列之间，则分别存在不同数量的碱基差异。即MLST方法只能用于血清型之间，不能在血清型内部进行分型研究。结论 与MLST比较，PFGE方法在肠炎沙门菌的分型方面显示了比较高的分辨率。     

Flagellin gene polymorphism analysis of Campylobacter jejuni infecting man and other hosts and comparison with biotyping and somatic antigen serotyping.

Flagellin gene sequence polymorphisms were used to discriminate amongst 77 strains of Campylobacter jejuni from sporadic and outbreak-associated human enteric infections, and from chickens, sheep and calves. The results were assessed in relation to Lior biotyping and serotyping (Penner somatic antigens). Eight DNA PCR-RFLP patterns (genotypes) were identified by analysis of HinfI fragment length polymorphisms in flagellin gene (flaA) polymerase chain reaction (PCR) products. One genotype (F-1) was a feature of 55% of strains. Strains within the genotypes were heterogeneous with respect to somatic antigens with 12 serogroups represented amongst the C. jejuni isolates of flaA type F-1. Serogroups Pen 1, 2 and 23 were the commonest (45%) amongst the 20 different serogroups represented. Several unique clusters of isolates with diverse biotypes were defined, and one cluster (F-7/Pen 23) contained epidemiologically implicated outbreak strains as well as sheep and calf isolates. We conclude that HinfI flaA typing is reproducible and offers high typability, and its combination with serogrouping provides a novel approach to characterizing isolates of C. jejuni with improved discrimination.     

Epidemiological characteristics ofPseudomonas aeruginosa strains causing infection in an Italian general hospital

During the 1989 calendar year,P. aeruginosa caused clinical infections in 0.46% of patients admitted to Ospedali Riuniti (a general hospital), Bergamo, Italy. Strains (n=267) ofP. aeruginosa were collected during this period, and epidemiological characteristics were studied. The mean prevalence ofP. aeruginosa infection in inpatients was 1.1% (range 0.06–7.3), whereas outpatients showed a significantly lower prevalence of infection (0.05%). Strains were recovered from inpatients of surgical wards (n=126; 47.2%), and outpatients (n=15; 5.6%). Males were more often affected than females (2.7:1). Infection of the urinary tract was the most common (34.1%).Pseudomonas aeruginosa was also involved in lower respiratory tract infections (18.7%) and septicaemia (17.6%). Four typing methods were performed, i.e. serotyping, antibiotyping, pyocin typing, and restriction endonuclease analysis (REA). Serotypes 0:11 and 0:6 were endemic in the hospital. Some serotypes correlated with specific clinical wards. Pyocin typing was an unreliable epidemiological tool. However, antibiotyping showed the presence of some epidemic clusters, probably related to the antibiotic consumption of the patients. REA suggested the circulation of endemicP. aeruginosa strains in both the obstetrics and neurosurgery wards.     

Australian multicentre comparison of subtyping methods for the investigation of Campylobacter infection

In order to identify subtyping methods able to contribute to the surveillance or investigation of Australian Campylobacter infection, six genotypic and three phenotypic subtyping methods were evaluated on a collection of 84 clinical isolates collected over a 30-month period from one region in Australia. The aim was to compare the logistics of various subtyping methods and examine their ability to assist in finding outbreaks or common sources of sporadic infection. The genotypic subtyping methods used were sequencing of the short variable region of the flaA gene, two methods using restriction fragment length polymorphism (RFLP) of the flaA gene using either DdeI or EcoRI with PstI, automated ribotyping, pulsed field gel electrophoresis and multilocus sequence typing. The phenotypic methods employed included Laboratory of Enteric Pathogens serotyping, Lior biotyping and antibiotic resistotyping. The level of agreement between subtyping results was determined. Phenotypic methods showed little agreement whereas genotypic typing methods showed a high level of agreement. Using the premise that five of the six genotypic typing methods were in agreement 15 genotypic groupings were identified. Sequencing of the short variable region of the flaA gene, RFLP of the flaA gene or automated ribotyping in conjunction with multilocus sequence typing best identified genotypic groupings. An alternative combination of RFLP of the flaA gene followed by ribotyping was equally satisfactory. RFLP of the flaA gene appeared to be suitable as a preliminary typing method based on ease of operation, equipment availability and cost.     

Molecular epidemiological investigation of an outbreak of Campylobacter jejuni identifies a dominant clonal line within Scottish serotype HS55 populations.

Three molecular typing methods, pulsed-field gel electrophoresis (PFGE), ribotyping, and flagellin (flaA) gene typing, were used to discriminate within a group of 28 Campylobacter jejuni, heat-stable serotype 55 (HS55) isolates derived from cases of campylobacter enteritis occurring throughout Scotland, including 9 isolates associated with an outbreak. PFGE was found to be most discriminatory, identifying 6 distinct profiles, followed by ribotyping (5 profiles), and then flagellin gene typing (4 profiles). The coincidence of all three genotypic markers identified a dominant clonal line within the HS55 group, accounting for each of the outbreak strains, and for 9 of the 19 sporadic strains. A second, closely related, clonal line accounted for a further 5 of the sporadic strains, and also included the HS55 reference strain. Identification and monitoring of such clonal lines should facilitate more effective future epidemiological surveillance of C. jejuni.     

Molecular typing of Aspergillus terreus isolates by random amplification of polymorphic DNA

Forty three isolates of Aspergillus terreus of environmental or clinical origin were typed by random amplification of polymorphic DNA (RAPD) with two different primers NS3 and NS7 from the fungal ribosomal 18S subunit gene. For the 31 epidemiologically unrelated isolates tested, the primers NS3 and NS7 gave rise to 23 and 24 different genotypes, respectively, and combining the results obtained with the two primers allowed the differentiation of all these isolates. No clustering was found in relation to pathogenicity, clinical signs, or geographic origin of the isolates. Five groups of related isolates of A. terreus were also typed. Analysis of sequential isolates from patients with cystic fibrosis or with invasive aspergillosis showed the clonality of the colonization or infection by A. terreus. Likewise, this straightforward typing method demonstrated the clonal origin of a massive contamination of the environment in a haematology unit. Therefore this RAPD typing method may constitute a valuable tool for the epidemiological follow-up of airway colonization in patients with cystic fibrosis or investigations of links between nosocomial outbreaks of invasive aspergillosis and environmental contamination.     

Comparative study of three different DNA fingerprint techniques for molecular typing of Shigella flexneri strains isolated in Romania

In this study, 97 epidemiologically unrelated Shigella flexneri strains isolated during 1994 (69 isolates) and 1997 (28 isolates) were characterised by ribotyping, enterobacterial repetitive intergenic consensus sequence-based PCR typing, and pulsed-field gel electrophoresis. Number of strains belonging to each of the six serotypes is selected equal to their distribution in Romania. The isolates comprise 24 ribotypes based on combination of two restriction patterns obtained with HindIII and PstI, respectively, 7 enterobacterial repetitive intergenic consensus (ERIC)-PCR types, and 92 XbaI pulsed-field gel electrophoresis (PFGE) patterns grouped in 31 pulsotypes at Dice coefficients of 85% similarity. We find no significant difference in the distribution of isolates collected during the two periods. Macrorestriction analysis by PFGE offers maximal discrimination. There seems to be little genetic variability among circulating S. flexneri strains of serotype 2a, suggesting that even a combination of several molecular techniques, including PFGE, could not easily differentiate an outbreak strain from temporally associated independent isolates.     

Pediatric parapneumonic empyema, Spain

Pediatric parapneumonic empyema (PPE) has been increasing in several countries including Spain. Streptococcus pneumoniae is a major PPE pathogen; however, antimicrobial pretreatment before pleural fluid (PF) sampling frequently results in negative diagnostic cultures, thus greatly underestimating the contribution of pneumococci, especially pneumococci susceptible to antimicrobial agents, to PPE. The study aim was to identify the serotypes and genotypes that cause PPE by using molecular diagnostics and relate these data to disease incidence and severity. A total of 208 children with PPE were prospectively enrolled; blood and PF samples were collected. Pneumococci were detected in 79% of culture-positive and 84% of culture-negative samples. All pneumococci were genotyped by multilocus sequence typing. Serotypes were determined for 111 PPE cases; 48% were serotype 1, of 3 major genotypes previously circulating in Spain. Variance in patient complication rates was statistically significant by serotype. The recent PPE increase is principally due to nonvaccine serotypes, especially the highly invasive serotype 1.     

Molecular typing by amplified fragment length polymorphism and PCR-restriction fragment length polymorphism,biotyping and antimicrobial susceptibility of Campylobacter jejuni

Molecular typing systems have provided invaluable information for tracking infectious agents through the food chain. These tools have been essential for understanding the epidemiology of gastrointestinal infectious diseases, therefore providing essential and evidence-based information for appropriate interventions and preventative measures. Two such molecular typing techniques based on the polymerase chain reaction (PCR) that have been applied to the epidemiology of foodborne pathogens are, amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) analysis. Campylobacter is responsible for one of the most common bacteria foodborne gastrointestinal infections affecting humans, especially in developed countries. The object of this paper is to apply AFLP and RFLP analysis of the flagellin (flaA) gene to 18 isolates of Campylobacter jejuni from human sporadic cases in Italy. Results of these analyses were compared to the phenotypes of these isolates based on biotyping and antimicrobial resistance determinations. All isolates were typable by the four methods. The RFLP procedure was performed with DdeI and HinfI enzymes, and 12 and 8 distinct profiles respectively were recognised. AFLP analysis was more discriminatory, and recognised 16 different profiles. Results from AFLP were reproducible and applicable for definitive characterisation of C. jejuni isolated from different outbreaks. PCR-RFLP of the flaA gene represents a useful tool only to compare isolates within a single outbreak.     

Comparison of molecular markers for strain typing of Leishmania infantum

The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles (kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV- patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.     

儿童侵袭性肺炎链球菌感染的血清型和耐药性

目的 了解某医院住院患儿侵袭性肺炎链球菌感染的临床特征以及菌株的血清型和耐药性,以期指导临床合理用药,寻找防治侵袭性肺炎链球菌感染的有效方法.方法 回顾性分析2014年1月—2018年12月该院明确诊断为侵袭性肺炎链球菌感染患儿的临床资料,肺炎链球菌的药敏结果、血清型,以及疫苗对其血清型覆盖情况.结果 74例侵袭性肺炎...     

Investigation of classical epidemiological links between patients harbouring identical, non-predominant meticillin-resistant Staphylococcus aureus genotypes and lessons for epidemiological tracking

According to molecular epidemiology theory, two isolates belong to the same chain of transmission if they are similar according to a highly discriminatory molecular typing method. This has been demonstrated in outbreaks, but is rarely studied in endemic situations. Person-to-person transmission cannot be established when isolates of meticillin-resistant Staphylococcus aureus (MRSA) belong to endemically predominant genotypes. By contrast, isolates of infrequent genotypes might be more suitable for epidemiological tracking. The objective of the present study was to determine, in newly identified patients harbouring non-predominant MRSA genotypes, whether putative epidemiological links inferred from molecular typing could replace classical epidemiology in the context of a regional surveillance programme. MRSA genotypes were defined using double-locus sequence typing (DLST) combining clfB and spa genes. A total of 1,268 non-repetitive MRSA isolates recovered between 2005 and 2006 in Western Switzerland were typed: 897 isolates (71%) belonged to four predominant genotypes, 231 (18%) to 55 non-predominant genotypes, and 140 (11%) were unique. Obvious epidemiological links were found in only 106/231 (46%) patients carrying isolates with non-predominant genotypes suggesting that molecular surveillance identified twice as many clusters as those that may have been suspected with classical epidemiological links. However, not all of these molecular clusters represented person-to-person transmission. Thus, molecular typing cannot replace classical epidemiology but is complementary. A prospective surveillance of MRSA genotypes could help to target epidemiological tracking in order to recognise new risk factors in hospital and community settings, or emergence of new epidemic clones.     

Efficient Differentiation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen’s University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter–Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a “gold standard” method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.     

Distribution of mecA among methicillin-resistant clinical staphylococcal strains isolated at hospitals in Naples,Italy

Two hundred and twenty strains of Staphylococcus isolated in Naples, Italy, were surveyed for the distribution of the mecA, the structural gene for penicillin-binding protein 2a, which is the genetic determinant for methicillin-resistance in staphylococci. Screening by a cloned mecA, revealed that of 220 strains, 43 were methicillin-resistant (19.5%) and 177 were methicillin-susceptible (80.5%). Among the 43 resistant strains 23 (53.5%) carried mecA in their genome and 20 (46.5%) did not carry mecA, in spite of their resistance to methicillin. Every group was submitted to the AP-PCR profiling. A quantitative analysis of the patterns divided strains into four different clusters for methicillin-resistant mecA-negative and two different clusters for methicillin-resistant mecA-positive with primer 1, while no clusters were noted with primer 7. We conclude that these clinical isolates from our area, were not found to belong to a single clone, although the predominance of four methicillin-resistant mecA-negative genotypes were noted.     

Pneumococcal meningitis in Greece: A retrospective serotype surveillance study in the post-PCV13 era (2010–2020)

BackgroundAs Greece is a country which has introduced the 13-valent pneumococcal conjugate vaccine (PCV13) both in the infant and in the adult immunization programs, the aim of the study was to investigate age-specific and serotype-specific trends of pneumococcal meningitis over an 11-year period (2010–2020).Materials and MethodsData are reported from pneumococcal meningitis cases [notified to the National Public Health Organization (NPHO)], with clinical samples and bacterial isolates sent for pneumococcal identification and serotyping at the National Meningitis Reference Laboratory (NMRL). Pneumococcal identification was performed directly on clinical samples or bacterial isolates by multiplex PCR (mPCR) assay, while serotyping was carried out by application of the Capsular Sequence Typing (CST) method with the combination of single tube PCR assays.ResultsA total of 427 pneumococcal meningitis cases were notified to the NPHO between 2010 and 2020. Among those, 405 (94.8%) were microbiologically confirmed, while samples from 273 patients were sent to the NMRL for identification and/or further typing. The annual notification rate peaked at 0.47/100,000 in 2016 and since then has been decreasing. The incidence was highest in infants and in older adults. Pneumococcal serotypes were identified in 260/273 (95.2%) cases, where clinical samples were sent to the NMRL. The most prevalent serotypes (≥5%) were 3, 19A, 23B, 15B/C, 11A/D, 23A, 22F. During the study period there has been a decrease of PCV13 serotypes combined with an increase of non-PCV13 serotypes (p = 0.0045).ConclusionsThis is the first study to report serotypes for pneumococcal meningitis across all ages in the post-PCV13 era in Greece. There is a need to enhance surveillance, by close monitoring of the emerging serotypes and the impact of vaccination programs. Higher-valency PCVs may help to improve the coverage of pneumococcal disease.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

Comparison of multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA) and phage typing for typing of Listeria monocytogenes.

The discriminatory power of four methods for typing of Listeria monocytogenes was compared. The four methods were multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA), and a newly developed Danish phage typing system. Ninety-nine human clinical, food and slaughterhouse isolates of Listeria monocytogenes were typed by each method. The most discriminatory single typing method was phage typing with an overall discriminatory index (DI) of 0.88 followed by REA, MEE and ribotyping with DI-values at 0.87, 0.83 and 0.79 respectively. Considering strains from each of the two predominant O-serotypes alone, serotype 1 was best discriminated by the molecular typing methods, in particular REA, which showed a DI of 0.92. The serotype 4 strains were best discriminated by phage typing (DI = 0.78). If two or more typing methods were combined, the combination of REA and MEE were found to be the most discriminatory combination. The DI values were 0.96, 0.74 and 0.90 for serotype 1, 4, and both combined, respectively. Phage typing is a rapid and inexpensive typing method but not as reproducible as the molecular typing methods. It is the most suitable method for mass screening. In situations where results are required to be highly reliable, i.e. when studying the relationships between only a few strains, a single or a combination of molecular typing methods should be used, preferable MEE and REA.