Characteristics of Listeria monocytogenes strains isolated in Russia and their typing using pulse electrophoresis

On the basis of specially developed scheme for the isolation of Listeria strains comprising 2 enrichment stages and the use of growth inhibitors, 128 L. monocytogenes cultures were isolated from clinical material, foodstuffs and sewage water. Highly virulent L.monocytogenes strains isolated from clinical material belonged to serovar 4b (54%) and 1/2a (38%), while those isolated from foodstuffs and sewage water belonged to 4b (74%). The restriction analysis of the chromosomal DNA of the isolated cultures with the use of restrictase EcoR1 on the basis of pulsed-field gel electrophoresis (PFGE) made it possible to distinguish Listeria strains in accordance with 5 types of restrictograms. The restrictograms of highly virulent L. monocytogenes strains, serovar 4b, belonged to types 1 and 2, while those of L. monocytogenes strains, serovar 1/2a, belonged to types 2 and 3. The comparative use of different methods for typing L. monocytogenes (sero-, phago-, bio- and resistotyping, the analysis of plasmid composition and restriction analysis) revealed that the combination of serotyping and restriction analysis on the basis of PFGE proved to be most promising for the characterization of the isolated L. monocytogenes strains and the assessment of their epidemic importance.     

Inhibition of Salmonella intracellular proliferation by non-phagocytic eucaryotic cells

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone.     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

Cell wall teichoic acid glycosylation in Listeria monocytogenes serotype 4b requires gtcA, a novel, serogroup-specific gene

We have identified a novel gene, gtcA, involved in the decoration of cell wall teichoic acid of Listeria monocytogenes serotype 4b with galactose and glucose. Insertional inactivation of gtcA brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid. Interestingly, the composition of membrane-associated lipoteichoic acid was not affected. Complementation of the mutants with the cloned gtcA in trans restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within L. monocytogenes, sequences homologous to gtcA were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b, gtcA appears to be the first member of a bicistronic operon which includes a gene with homology to Bacillus subtilis rpmE, encoding ribosomal protein L31. In contrast to gtcA, the latter gene appears conserved among all screened serotypes of L. monocytogenes.     

Detection of "Staphylococcus aureau" protein A on selective culture media (author's transl)

Production of protein A by 100 various animal strains and 106 human strains of Staphylococcus aureus has been detected by conditioned haemagglutination after growth on seven different culture media: agar medium at n degrees 2 and Columbia agar with and without nalidixic acid (10.8 micrograms/ml), Baird-Parker, Chapman and tellurite-glycine-agar media. Rate of protein A-producing strains is as high on nalidixic acid media as on the same media without antibiotic. Baird-Parker, Chapman and tellurite-glycine-agar media are not convenient for protein A detection.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Disinfection of eyelid speculums for retinopathy of prematurity examination

Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924-1929, 1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes 1/2c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100 degrees C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.     

Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100 degrees C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Bovine mastitis caused by Listeria monocytogenes: kinetics of antibody responses in serum and milk after experimental infection

The kinetics of antibodies in serum and milk directed against proteins from Listeria monocytogenes were studied using 4 lactating cows after infection was experimentally induced in the udder with four strains of serotypes 4b or 1/2a. Antibodies (IgG and IgA) in samples of composite quarter milk and serum of the cow were measured by indirect ELISA. Microtiter plates were coated with proteins obtained from the culture supernatant of L. monocytogenes 4b. After challenge, an IgG response in serum and milk to listerial infections in the udder occurred for all cows, although the response varied among cows. In sera, the IgG titers reached a peak at 9 to 13 wk after challenge and remained elevated until 21 to 33 wk after challenge. In milk, the IgG titer increased significantly 3 wk after the challenge for all cows. A weak and nonpersistent increase in IgA antibodies also occurred. These results indicate that IMI by L. monocytogenes induced an increase of antibodies in milk, which could be detected with an ELISA test using our antigenic preparation. Therefore, this antigenic preparation could be used for the evaluation of a new method of diagnosis for bovine mastitis caused by L. monocytogenes.     

Epithelial tumors with thymus differentiation of the thyroid gland and the neck

A total of 1100 samples of prepared foods purchased in restaurants and delicatessen shops in the city of Barcelona was examined for the presence of Listerio. L. monocytogenes was more frequently isolated from foods intended to be consumed without further cooking (9.3%) than from foods intended to receive further cooking prior to consumption (2.9%). A quantitative study, carried out with 773 samples, yielded 1% of samples with numbers of L. monocytogenes higher than 100 CFU/g. Strains of L. monocytogenes belonged to serovar 4b (36.3%), 1/2a (29.5%), 1/2b (22.7%) and 1/2c (11.3%). L. monocytogenes serovar 4b phagevar 3274: 2671 (6 of 11) was prevalent among the strains studied.     

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule

Listeria monocytogenes blood agar (LMBA) was compared to Listeria selective agar based on lithium chloride and ceftazidime (LA) and to the Oxford and Palcam media recommended by ISO and IDF for the detection and enumeration of L. monocytogenes from foodstuffs and food-processing environments. LMBA is based on trypticase soy agar with the following additions: sheep blood (5%) and as selective agents lithium chloride (10 g/l), polymyxin B sulphate (10 mg/l) and ceftazidime (20 mg/l), whereas the selectivity of LA is based on lithium chloride (15 g/l) and ceftazidime (15 g/l). The media were compared in the detection of L. monocytogenes after enrichment from naturally contaminated foodstuffs (n = 423) and from food-processing environments (n = 93), and in the enumeration of the species from naturally contaminated foodstuffs (n = 287). LMBA was superior both to the standard media and to LA in detection after enrichment and also in enumeration, except in the case of fresh broiler cut samples. The overall sensitivities of the Palcam, Oxford, LA and LMBA media were 68%, 67%, 74% and 96% in detection after enrichment and 64%, 73%, 76% and 80% in the enumeration of the species from ready to eat foods. The superiority of LMBA is based on distinguishing L. monocytogenes from other Listeria species by detection of beta-hemolysis, whereas the other media gave false-negative results because of the overgrowth of Listeria spp. other than L. monocytogenes, especially in detection after enrichment. A more selective medium than LMBA would have been required for the enumeration of the species from samples with high levels of competitive bacteria other than Listeria spp. The results indicate the need for a more specific isolation medium for L. monocytogenes in addition to those recommended by ISO and IDF for both detection and enumeration. LMBA offers an alternative to be used in combination with either Palcam or Oxford as well as with LA.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Association of enterohemorrhagic Escherichia coli hemolysin with serotypes of shiga-like-toxin-producing Escherichia coli of human and bovine origins

In this study we investigated whether the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene ehxA could be used as an indicator of pathogenicity in Shiga-like-toxin-producing Escherichia coli (SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence of ehxA and the virulence-associated genes eae, slt-I, and slt-II. The percentages of human isolates in groups 1 and 2 that were positive for ehxA were 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for ehxA were 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive for eae were 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for eae were 78, 15, and 19%, respectively. The frequencies of both ehxA and eae were significantly higher for group 1 isolates than for group 2 isolates. The presence of the ehxA gene was associated with serotype, as was the presence of the eae gene. Some serotypes, such as O117:H4, lacked both eae and ehxA and have been associated with severe disease, but only infrequently. The slt-I genes were more frequent in group 1 isolates than in group 2 isolates, and the slt-II genes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of the ehxA and slt genes in E. coli isolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. Eight E. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1, 080 E. coli isolates were examined, and the ehxA gene was detected in 12 independent strains, only 3 of which were positive for slt. We concluded that the ehxA gene was less correlated with virulence than the eae gene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of the ehxA gene in bovine isolates that are not SLTEC.     

Incidence of Listeria monocytogenes in cheese produced in Rio de Janeiro, Brazil

The present study evaluated the incidence of Listeria spp. in some Brazilian cheeses obtained from retail stores in Rio de Janeiro, Of 103 samples of various types of cheese examined as recommended in the Listeria isolation protocol of the Health Protection Branch of Canada, 11 (10.68%) were contaminated by Listeria monocytogenes, 13 (12.62%) by Listeria innocua, 6 (5.83%) by Listeria grayi, and 1 (0.97%) by Listeria welshimeri. A higher incidence of L. monocytogenes as observed mainly in the homemade Minas Frescal cheeses (a Brazilian soft white cheese, eaten fresh), 7 of 17 (41.17%), followed by ripened cheeses, 3 of 53 (5.67%), and industrially manufactured Frescal (Minas and Ricotta) cheeses, 1 of 33 (3.03%). Three serotypes (1/2a, 1/2b and 4b) were observed among the strains of L. monocytogenes isolated, all of them being frequently involved in outbreaks of foodborne listeriosis and sporadic cases of the disease all over the world.     

Isolation of Listeria monocytogenes in slaughter animals and bats of Togo (West Africa)

Listeria monocytogenes was isolated from the intestinal lymph nodes of 8 out of 342 apparently healthy slaughter animals at the municipal abattoir in Lome (Togo) and from the faeces of 3 out of 236 bats. Except two strains of serovar 4b isolated from bats, all the nine others belonged to serovar 1/2a. The positive findings of slaughter animals were made from the carcasses of 2 pigs, 5 little ruminants and 1 cattle. The findings are discussed in the light of present knowledge on the occurrence of L. monocytogenes in some species of animals and its implications for the human being.     

Heat resistance of Listeria monocytogenes by two recovery media used with and without cold preincubation

Three strains of Listeria monocytogenes were heat-treated at three temperatures in physiological saline by a capillary tube method. Recovery of heat-treated bacteria was performed on blood agar and on tryptose phosphate agar with ferric citrate and aesculin (TPA-FE). Both media were used in two ways: (1) incubation at 37 degrees C for 7 d, and (2) preincubation at 4 degrees C for 5 d in order to obtain repair of heat-injured bacteria, followed by incubation at 37 degrees C for 7 d. D and z values were determined. In both incubation procedures, better average recovery was obtained on blood agar than on TPA-FE. Thus, higher D values were recorded when blood agar was used. In most cases the differences were statistically significant. Repair at 4 degrees C of heat-injured bacteria occurred on both media but the proportions of repaired bacteria were higher on blood agar. The repair on this medium was generally reflected in higher D values for preincubated samples. Some significant differences in heat resistance were noted between the strains.     

Rapid determination of Listeria monocytogenes in foods using a resuscitation/selection/kit system detection

A resuscitation medium consisting of a trypticase soy broth base supplemented with 0.5% yeast extract, 0.25% sodium pyruvate, 0.01% sodium thioglycollate, and 0.1% chicken fat was used in the resuscitation of heat-injured and freeze-injured cells of Listeria monocytogenes. After a resuscitation period of 4-h, the medium was made selective through the addition of nalidixic acid, acriflavin, and cycloheximide. The organisms were incubated in the selectivized medium at 35 degrees C for an additional 16 h. The numbers of resuscitated Listeria monocytogenes cells rose from 10(1) to 10(7) cells/mL in 20 h. Similar numbers of Staphylococcus aureus, Escherichia coli, and Salmonella bonn were grown together with Listeria monocytogenes; these organisms did not inhibit the growth of Listeria monocytogenes nor interfere with its detection by the Listeria-Tek kit system. The resuscitation/selection/kit system (RSK) was compared with the methodology in the Bacteriological Analytical Manual (BAM) for the detection of Listeria monocytogenes in 22 naturally contaminated cheese samples: 8 of these were positive by the BAM system and 12 were positive by the RSK system. The 8 Listeria positives found by the BAM system were positive by the RSK system. All 12 Listeria-presumptive positive samples by the RSK system were confirmed to be Listeria monocytogenes. The use of the RSK system enhanced the recovery of the pathogen, and detection was accomplished within 24 h.     

The chemical stability of enkephalin-like tetrapeptides in a cannula implanted in the rat neostriatum for multiple microinjections

A method for identification of Listeria in food samples was developed. It consisted of cultivating of suspected specimen on standard agar medium, direct absorption of grown colonies onto nitrocellulose membrane and processing of the latter with rabbit serum raised against purified cell wall protein Lm79/39 of L. monocytogenes. Analysis using anti-rabbit peroxidase conjugate and 4-chloro-1-naphthol and H2O2 solutions allowed direct detection of Listeria colonies which remained readily available for subsequent isolation.     

eaeA genes in Escherichia coli derived from Japanese patients with sporadic diarrhea

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: O26:H-(2/3), O55:H7 (4/4), O55:H-(2/ 2) and O128:H2(3/3). Two enterohemorrhagic E. coli (EHEC) strains (Verotoxin positive O157:H7) were also eaeA positive. Among 260 non-EPEC strains that were not categorized as diarrheagenic E. coli, 16 (6%) were eaeA positive. Those serotypes were as follows: O15:H2, O20:H6, O28:H28, O63:H6. O153:H7, O28:H6, O153:H19 and O157:H45. EPEC strains including O18:H7 and six other serotypes, enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC) were all eaeA negative.     

Xylose lysine deoxycholate agar for the isolation of Salmonella and Shigella from clinical specimens

Xylose lysine deoxycholate (XLD) agar was more efficient for the detection of Shigella sonnei than Salmonella-Shigella (SS) agar and Drigalski agar, XLD was slightly more efficient for the detection of Salmonellae by direct plating than SS agar. Xylose lysine deoxycholate agar is proposed as the second selective plating medium for Shigellae isolation. It is also a useful second selecting plating medium for the isolation of Salmonellae serotypes, which form non-characteristic colonies on Bismuth-Sulfite agar. Efficiency comparisons between 4 direct plating media and one enrichment medium were made during the period of 3 years by analysing 21793 fresh clinical stool specimens. In that time 1394 Salmonella and 519 Shigella strains were isolated.