Validation of NMKL method No. 136--Listeria monocytogenes, detection and enumeration in foods and feed.

A collaborative study was organised to define the performance characteristics of the revised NMKL Method No.136 "Listeria monocytogenes. Detection and enumeration in foods". Chromogenic L. monocytogenes specific plating medium, Agar Listeria according to Ottaviani and Agosti (ALOA) was introduced in the revised method in order to improve the sensitivity and specificity of the method, and to shorten the analysis time. Efficacy of ALOA One Day from AES (ready-to-use agar in bottles), Listeria Chromogenic Agar (Agosti and Ottaviani Listeria agar) from Lab M (LCA) (dehydrated powder), Chromogenic Listeria Agar Plates from Oxoid (OCLA) (ready-to-use plates) and L. monocytogenes blood agar medium LMBA from Lab M (dehydrated powder) were tested. Three types of food matrices (vacuum-packed hot-smoked salmon, soft cheese and cooked ham) and one feed matrix (wheat grain) inoculated with two levels of L. monocytogenes with or without L. innocua were used in the study. A total of 24 samples were analysed both in the detection and enumeration part of the study by 18 and 17 Nordic laboratories, respectively. The sensitivities of ALOA, LCA, OCLA and LMBA in the detection of L. monocytogenes in food samples after one-step enrichment (Half-Fraser) were 94.4-96.4% and after two-step enrichment (Half-Fraser followed by Fraser) 97.7-100%. For wheat grain the respective figures were 84.7-88.9% and 90.3-93.1%, respectively. The precision characteristics were generally good for the enumeration of L. monocytogenes in the food samples with high levels of inoculation. Several poor values obtained from the food samples with low levels of inoculation probably reflect high uncertainty of measurement when less than 10 cfu/g was counted. Poor values obtained from the wheat grain samples by any of the media evaluated were due to poor precision for feed samples. According to the study, the revised NMKL Method No.136, 4th ed. showed excellent results in the detection and satisfactory results in the enumeration of L. monocytogenes in foods. The results for the detection of L.monocytogenes in wheat grain were good, but the method cannot be recommended for the enumeration of L. monocytogenes in feed-stuffs. Any one of the media evaluated can interchangeably be used as an obligatory isolation medium for the detection and enumeration of L. monocytogenes in foods, and for the detection in feed-stuffs. The L. monocytogenes specific plating media that were evaluated shorten the time of analysis and significantly reduce the work load. The detection of positive samples mostly after Half-Fraser enrichment, reduces the analysis time further, and makes it possible to skip the secondary enrichment. However, secondary enrichment cannot be totally left out, because samples with low levels of L. monocytogenes, with high levels of competing flora, and with injured L. monocytogenes, do need secondary enrichment.

Bacteriological quality of organically grown leaf lettuce in Norway.

AIM: To investigate bacteriological quality in organically grown leaf lettuce, including the presence of selected pathogenic bacteria, and to obtain information about organic lettuce production, including fertilizing regimes. METHODS AND RESULTS: Altogether 179 samples of Norwegian organically grown lettuce were collected from 12 producers. Escherichia coli was isolated from 16 of the lettuce samples, but in 12 of these contamination was sufficiently low (<100 CFU g(-1)) that they would be considered to be of acceptable bacteriological quality. Escherichia coli O157 and Salmonella were not detected in any of the samples. Listeria monocytogenes serogroups 1 and 4 were isolated from two samples. CONCLUSIONS: Organic lettuce produced in Norway was generally of acceptable bacteriological quality, but the results show that contamination of organic lettuce with E. coli and L. monocytogenes do occasionally occur. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that organically grown lettuce may be contaminated with E. coli and L. monocytogenes during cultivation.

Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products.

AIM: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. METHODS AND RESULTS: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. CONCLUSIONS: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.

Validation of a PCR-based method for detection of food-borne thermotolerant campylobacters in a multicenter collaborative trial.

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.

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.

An outbreak of listeriosis suspected to have been caused by rainbow trout.

An outbreak of listeriosis in Sweden, consisting of nine cases, was investigated by means of molecular typing of strains from patients and strains isolated from suspected foodstuffs, together with interviews of the patients. Listeria monocytogenes was isolated from six of the patients, and all isolates were of the same clonal type. This clonal type was also isolated from a "gravad" rainbow trout, made by producer Y, found in the refrigerator of one of the patients. Unopened packages obtained from producer Y were also found to contain the same clonal type of L. monocytogenes. Based on the interview results and the bacteriological typing, we suspect that at least six of the nine cases were caused by gravad or cold-smoked rainbow trout made by producer Y. To our knowledge, this is the first rainbow trout-borne outbreak of listeriosis ever reported.

The treatment of traumatic facial bones and jaw fractures in the period 1972-1996.

During the last twenty-five years, in the period from January 1, 1972 to December 31, 1996, 1006 injured with facial and jaw fractures have been treated at the Clinic for Maxillofacial Surgery of the Military Medical Academy--91.05% of the injured were males and 67.98% belonged to the age group between 11 and 30. The most frequent etiologic factors were traffic accidents (46.42%) and fights (38.07%). Isolated fractures of mandible were found in 51.09% of the injured. In 70.57% of the treated the surgery was used for adequate reduction, osteosynthesis and stabilization of bone fragments, and 29.34% of the injured were treated conservatively, which involved the manual reduction and intermaxillar immobilization. The post-operative complications were observed in 51 (5%) of the injured.

Vascularized fibular graft in the reconstruction of posttraumatic mandibular defects.

The results of the use of the vascularized fibular graft for the reconstruction of posttraumatic defects of the mandible are presented. Different surgical procedures were applied in reconstruction of the mandibular defects in 120 wounded. In the last four years vascularized fibular graft was used in 11 mandibular reconstructions. Taking into account the survival rate of 100%, long vascular pedicle, strength, length and the possibilities of adaptation of the bone make this graft suitable for mandibular reconstruction. The functional and esthetic results obtained by the use of this graft were satisfactory, with minimal donor site morbidity.

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 patient's 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 patient's refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.

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

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.

Prevalence of Listeria monocytogenes and other Listeria spp.in smoked and 'gravad' fish.

Altogether 150 samples of vacuum-packed fish were examined for the presence of Listeria species. Listeria monocytogenes were isolated from 12 of 58 'gravad' fish samples, 3 of 26 cold-smoked and one of 66 hot-smoked fish samples. Ten of these 16 positive samples harboured more than 100 L. monocytogenes cfu/g. The highest level (132,000) was found in a sample of hot-smoked rainbow trout (Oncorhynchus mykiss). Serogroup 1/2 was most frequently found, followed by 4 and 3. One sample of gravad rainbow trout harboured more than one serogroup of L. monocytogenes. L. innocua and L. seeligeri were isolated from 12 and 1 samples, respectively.

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.

Occurrence of Listeria species in broilers pre- and post-chilling in chlorinated water at two slaughterhouses.

Altogether 323 pooled samples of neck skins from 1615 broilers from 2 processing plants (A and B) were examined for the presence of Listeria species. The broilers were sampled pre-chilling-after leaving the final rinser but before entering the chiller with chlorinated water-and post-chilling-immediately upon leaving the chiller. Free available chlorine in the chilling water varied from 2 to 15 ppm in plant A and was about 10 ppm in plant B. Listeria monocytogenes was only isolated from broilers in plant A sampled post-chilling (58% of 62 samples). L. innocua was isolated from 19% and 39% of broilers sampled pre-chilling in plants A and B, respectively. Post-chilling, L. innocua was isolated from 3% and 6% of samples from plants A and B, respectively.

[Motivation in patients for surgical treatment of prognathism using bimaxillary surgery]. / Motivisanost pacijenata za hirursko lecenje progenije primenom bimaksilarne hirurgije.

A postoperative survey was conducted to evaluate the motivation of twenty three patients following surgical correction of mandibular prognathism. The operative technique of bimaxillary surgery was applied to all patients. The major reason for surgery was esthetics. Majority of patients were informed about surgery by their family dentists, and almost all of them were satisfied and stated that their expectations were fulfilled.

Occurrence of Listeria species in an abattoir for cattle and pigs in Bosnia and Hercegovina.

Altogether 496 samples of meat, lymph nodes, process water and swabs from different places in the abattoir were examined for the presence of Listeria spp. L. monocytogenes was isolated from 31 (6%) and other Listeria spp. from 65 (13%) samples L. monocytogenes was isolated from 2 of 10 beef meat samples, 4 of 50 pig meat samples and 1 of 21 lymph nodes of pigs. No Listeria bacteria were isolated from lymph nodes of cattle. The highest percentage of Listeria was recovered from the unclean sections (cattle 22% and pigs 27%) and the highest frequency was observed during the winter months.