Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

INFLUENCE OF TEMPERATURE ON THE FATTY ACID PROFILE OF LISTERIA MONOCYTOGENES

Variation in the fatty acid profile of two Listeria monocytogenes strains grown at varying temperatures was determined. The fatty acid profiles varied greatly at different temperatures. General decreases in relative percentages of branched and medium chain (up to C16:0) fatty acids and variable changes in long chain fatty acids were found with increasing growth temperature. Individual fatty acid percentages between strains were variable. The relative percentages of unknown long chain fatty acids, detected in both strains at various temperatures, were greatest in Scott A (7.07%) and ATCC 19114 (13.15%) at 35C. Results demonstrated that L. monocytogenes had altered fatty acid profile in response to changes in growth temperature.     

ENUMERATING LISTERIA MONOCYTOGENES IN FOOD: A PROBLEM OF LOW NUMBERS

Listeria monocytogenes is recognized as a serious foodborne pathogen in humans. However, an adequate enumeration method is still lacking for the examination of food products which are usually contaminated at low levels, < 100 CFU g⁻¹. The improvement of Listeria enumeration has given place to a considerable sum of research tasks, leading to the proposal of several alternative methods. Several attempts to enumerate L. monocytogenes with the most probable number technique or with methods based on molecular biology or bacteria concentration techniques have been reported. The objective of this paper is to synthesize the current knowledge concerning L. monocytogenes enumeration, by focusing particularly on the problem of the enumeration of low numbers.     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

DETERMINATION AND SIGNIFICANCE OF GENETIC DIVERSITY IN LISTERIA ISOLATED FROM FOOD AND ENVIRONMENT

Listeriosis in humans and animals is caused by the opportunistic bacterium Listeria monocytogenes. The repeated outbreaks in 1980s have increased the interest in the epidemiology of the disease. The organism is widely distributed in the environment, and healthy carriers are found among humans and animals. It has been isolated from a variety of foods including chicken, cheese, milk, sausages, and smoked, fermented and marinated fish products.
The ubiquity of L. monocytogenes and risk to the target populations make study of the epidemiology of the etiological agent important. Phage and serotyping have been employed in conjunction with other methods in modern molecular biology to isolate, identify and type the various strains of L. monocytogenes. Information regarding the genetic diversity among different species of the genus Listeria and among the same species is lacking. This report attempts to address this problem.     

RECOVERY and SPECIATION of LISTERIA FROM RAW and COOKED MEAT and POULTRY PRODUCTS

Recovery of Listeria from raw and cooked meat products was compared using Fraser broth (FB) enrichment incubated at 30 and 35C for 24 and 48 h. the Micro-ID Listeria test strip for biochemical characterization of Listeria was also compared with conventional tests. Listeria spp. were recovered from 33, 47, and 20 of the raw chicken, raw beef and cooked meat products, respectively. No false-negative reactions were observed and more total Listeria -positive samples were found using FB incubated for 48 h compared with 24 h. Samples incubated at 35C had fewer false negative tubes than those incubated at 30C. More false-positive FB tubes were observed after 48 h than after 24 h incubation. Over half of the cooked samples did not hydrolyze the esculin and turn the tubes black, and therefore did not have to be streaked onto selective plates. However, with raw chicken or beef because of the large number of false-positive FB tubes, almost all tubes had to be streaked onto selective plates and very little advantage was gained from using the FB. the Micro-ID Listeria test kit gave a 100% correlation with conventional biochemical reactions for pure cultures of Listeria isolated from the three categories of meat products in this study. When used in conjunction with hemolysis plates and CAMP reactions, this test identifies species of Listeria isolates within 24 h of visible colony formation.     

AN ELECTRICAL TESTING METHOD FOR THE DETECTION OF LISTERIA SPECIES IN CONFECTIONERY PRODUCTS

An electrical testing method for the detection of Listeria spp. in confectionery products and associated raw materials was developed in which samples can be negatively screened within 48h. The method involves a 24h resuscitation in Listeria enrichment broth followed by a 24h test in a Bactometer M128 using a broth (Claremont broth) developed from Oxford agar. The comparative study involved analysis of 511 samples (chocolate, dairy, cereal, nut and fruit products) tested by the Interim Australian Standard method (AS) and the Bactometer method (BM). The sensitivity and specificity of the BM for samples §1 Listeria/g was 100% and 99.8%, respectively, when compared to the AS method. When samples containing < 1 Listeria/g sample were added to the analysis, the sensitivity and specificity marginally dropped (98.3% and 99.6%). The electrical capacitance method is rapid and easy to perform, with a negative result being available within 48h making it a viable tool in a positive release QA program in food manufacturing factories.     

快速检测猪肉中单核细胞增多症李氏菌PCR试剂盒的研制

快速检测猪肉中单核细胞增多症李氏菌的ＰＣＲ试剂盒与分离培养法平行检测１５６份猪肉样品,结果ＰＣＲ的阳性率为５．８％,分离培养法的阳性率为３．８％,两者符合率为９７％。实验证明,该试剂盒具有良好的特异性和可重复性,可测出样品中至少３２ＯＣＦＵ细菌,并在２ｄ内取得结果,具有推广应用价值。     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY AND ENUMERATION OF SUBLETHALLY HEAT- AND ACID-INJURED LISTERIA MONOCYTOGENES

The suitability of PALCAM and modified Oxford (MOX) agars for recovering sublethally heat- and lactic acid-injured Listeria monocytogenes was investigated. L. monocytogenes LM101M, LM103M (meat isolates), and Scott A were suspended in tryptose phosphate broth (TPB), heated for up to 40 min at 54C, and surface plated onto tryptose phosphate agar (TPA), TPA + 4% NaCl (TPAS), PALCAM, and MOX. TPA and TPAS were used to determine total viable and sublethally injured populations, respectively. Heat-injured LM103M was recovered in the highest numbers on all media, followed by Scott A and LM101M (P<0.01). TPA allowed best recovery of all test strains, followed by PALCAMand MOX which were not different, and TPAS (P<0.01). For acid-injury studies, uninjured and heat-injured (54C for 20 min) test strains were suspended in phosphate-buffered TPB + 0.85% lactic acid (bTPBLA) at 25C for up to 24 h and plated as described above. Uninjured and heat-injured L. monocytogenes were recovered better from bTPBLA on MOX than on PALCAM (P<0.05). Heat injured L. monocytogenes LM103M was recovered better than LM101M but similar to Scott A on MOX and PALCAM (P<0.05), whereas Scott A was recovered similarly to LM101M and LM103M on MOX and PALCAM (P>0.05). Acid-injury of L. monocytogenes LM103M was enhanced by prior heat stress.