Semir Loncarevic

Modified Multiplex PCR Method for Detection of Pyrogenic Exotoxin Genes in Staphylococcal Isolates

ABSTRACT A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.

Occurrence of Listeria monocytogenes in soft and semi-soft cheeses in retail outlets in Sweden.

Samples of 333 retail cheeses produced in or imported into Sweden were examined for the presence of Listeria monocytogenes. Listeria monocytogenes was isolated from 6% of the cheese samples. Cheeses made from raw milk were more frequently contaminated with L. monocytogenes (42%) than cheeses made from heat-treated milk (2%). The incidence of the organism in whole cheeses and pre-cut wedges was similar (6%). L. monocytogenes was only found in imported cheeses (18 from France, and one from Germany and Italy, respectively). The numbers of L. monocytogenes varied from < 1 x 10(2) to 1 x 10(5) cfu/g. All L. monocytogenes strains belonged to serogroup 1/2, except isolates from two samples that belonged to serogroup 4.

Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish.

The first lesson learned from this outbreak was that vacuum-packed rainbow trout is not only an excellent medium for the growth of Listeria monocytogenes, but may also cause human listeriosis. Another lesson is that one single fish processing plant may spread multiple clonal types of L. monocytogenes by selling contaminated products to consumers. Thus, when investigating fish-borne outbreaks of listeriosis one should identify and type several isolates of L. monocytogenes from each food and environmental sample, since multiple clonal types might be present. The outbreak described in this paper involved at least eight human cases, three clonal types of L. monocytogenes, and lasted for 11 months. During the outbreak investigation, L. monocytogenes was also isolated from another brand of rainbow trout found in the refrigerator of one of the patients. These latter isolates belonged to a clonal type not associated with the outbreak. However, this clonal type is of considerable interest since it has been associated with foodborne outbreaks of listeriosis in several countries, and is also the second most common clonal type among human clinical isolates of L. monocytogenes in Sweden. Besides the described outbreak, it is likely that vacuum-packed, cold-smoked and gravad rainbow trout have been involved in additional cases of foodborne listeriosis in Sweden.

Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

The clones of Listeria monocytogenes detected in food depend on the method used

S. LONCAREVIC, W. THAM AND M.‐L. DANIELSSON‐THAM. 1996. Restriction enzyme analysis (REA) with pulsed‐field gel electrophoresis (PFGE) has been used to characterize and compare Listeria monocytogenes strains isolated from foods by two methods, an enrichment procedure and a direct plating procedure. In total 151 isolates from nine foods were investigated. In six of the foods (101 strains investigated) only one clone of L. monocytogenes was found irrespective of the method used. In three foods (50 strains investigated) the direct plating procedure yielded more clones than the enrichment procedure. At the most, five clones were detected in the same food. The results presented here indicate that direct plating from the food reveals more L. monocytogenes clones than revealed by an enrichment procedure.

A case of foodborne listeriosis in Sweden.

A 70‐year‐old woman fell seriously ill overnight with meningitis and was admitted to hospital. Cerebrospinal fluid culture yielded Listeria monocytogenes. One of the first problems in solving a human case of listeriosis suspected to be foodborne is to find the foods likely to have been transmitting L. monocytogenes. Two enrichment procedures and a direct plating procedure were used for isolation of the bacteria from different food items collected from the patients refrigerator, local retail store and producer. Samples of vacuum‐packed products of sliced pork brawn, sliced cooked medwurst and berliner wurst of the same brand harboured L. monocytogenes. Serotyping and restriction enzyme analysis (REA) with pulsed‐field gel electrophoresis (PFGE) were used to characterize and compare 41 isolates, including the human strain. At least three clones were present in the foods investigated, and one of these was identical to the human clone. This clone was present in samples of medwurst from the patients refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.

A rapid differentiation of Listeria monocytogenes by use of PCR-SSCP in the listeriolysin O (hlyA) locus

Single strand conformation polymorphism of PCR-amplified fragments (PCR-SSCP) was employed to develop a typing protocol for Listeria monocytogenes. Twelve sets of PCR primers were designed to amplify fragments within the coding and non-coding region of hlyA locus. In parallel, PFGE analysis of ApaI and AscI digested L. monocytogenes DNA was performed to determine the number of different genotypes and distribution of strains within serovar-subgroups. SSCP analysis of PCR generated amplicon K9 derived from the non-coding region of the hlyA gene revealed reproducible and highly polymorphic patterns whereas other amplicons showed either monomorphic or 2 to 6 different patterns. Combining the results of all 12 primer pairs, 25 genotypes were observed in 39 strains representing seven serovars. Results were confirmed by PFGE typing, only two differences in the contribution to subgroups in serovar 3b strains were observed. The data substantiate that the PCR-SSCP analysis is a reliable and highly discriminating method for characterizing L. monocytogenes strains on the molecular level.