Annukka Markkula

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

ABSTRACT Botulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2 spore/g for types A, B, and F to 10−1 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants

Persistent and non-persistent Listeria monocytogenes strains were tested for initial resistance and adaptive and cross-adaptive responses towards two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride and n-alkyldimethyl ethylbenzyl ammonium chloride, one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl, sodium hypochlorite and potassium persulphate. The initial resistance of two persistent and two non-persistent L. monocytogenes strains was observed to differ. Both types of strains adapted after a 2-h sublethal exposure to the quaternary ammonium compounds and the tertiary alkylamine, the highest increase in the minimum inhibitory concentration (MIC) being 3-fold. Progressively increasing disinfecting concentrations at 10 and 37 degrees C resulted in adaptation of L. monocytogenes to all disinfectants except potassium sulphate. The highest observed increase in MIC was over 15-fold, from 0.63 to 10 microg/ml of n-alkyldimethyl ethylbenzyl ammonium chloride. All strains reached approximately similar MICs. Stability of the increased resistance was tested by measuring MICs every seventh day for 28 days. The increased resistance to sodium hypochlorite disappeared in 1 week, but the quaternary ammonium compounds and the tertiary alkylamine showed increased resistance for 28 days. These results suggest that cellular changes due to adaptive responses continue to have an effect on the resistance some time after the exposure. All disinfectants were shown to cause cross-adaptation of L. monocytogenes, the highest increase in MIC being almost 8-fold. The only agent that L. monocytogenes could not be shown to cross-adapt to was potassium persulphate which did, however, cause cross-adaptation to the other disinfectants. The mechanism behind these adaptive responses seemed to be non-specific as cross-adaptation was observed not only between related but also unrelated disinfectants. These findings suggest that sustaining high disinfectant effectiveness may be unsuccessful by rotation, even when using agents with different mechanisms of action.

Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis

A total of 257 raw fish samples at two different sites were examined for the presence of Listeria monocytogenes. The prevalence of L. monocytogenes was 4%. From 11 positive samples, nine different L. monocytogenes pulsed-field gel electrophoresis genotypes were recovered. From nine pulsotypes recovered from raw fish and 32 pulsotypes shown by 101 fish product isolates, two raw fish and fish product pulsotypes were indistinguishable from each other. Although the prevalence of L. monocytogenes in raw fish is low, the range of L. monocytogenes strains entering the processing plant in large amounts of raw material is wide. This indicates that the raw material is an important initial contamination source of L. monocytogenes in fish processing plants. This postulation is supported by the identical pulsotypes recovered from both raw and processed fish. Some L. monocytogenes strains entering a plant may thus contaminate and persist in the processing environment, causing recurrent contamination of the final products via processing machines.

Genes encoding putative DEAD-box RNA helicases in Listeria monocytogenes EGD-e are needed for growth and motility at 3°C.

Quantitative RT-PCR revealed that transcripts of all four putative DEAD-box RNA helicase genes of the psychrotrophic pathogen Listeria monocytogenes EGD-e are found at higher levels in organisms grown at 3°C than at 37°C. At 3°C, growth of the three corresponding gene deletion mutants Δlmo0866, Δlmo1450 and Δlmo1722 was clearly restricted. The minimum growth temperatures of the three mutants were also higher than that of the wild-type EGD-e. In addition to inability to grow at 3°C, growth of Δlmo0866 and Δlmo1722 was reduced at 25°C, suggesting special roles of Lmo0866 and Lmo1722 in growth at suboptimal temperatures. Growth of Δlmo1450 was restricted not only at 3°C and 25°C, but also at 37°C, suggesting that Lmo1450 plays a universal role in growth of L. monocytogenes EGD-e. Moreover, cold-sensitive Δlmo0866, Δlmo1450 and Δlmo1722 were impaired in motility. The Δlmo0866 and Δlmo1450 strains were non-motile, while Δlmo1722 showed reduced motility. This study shows that the putative DEAD-box RNA helicase genes lmo0866, lmo1450 and lmo1722 are necessary for cold tolerance and motility of L. monocytogenes EGD-e.

Prevalence and Genetic Diversity of Listeria monocytogenes in the Tonsils of Pigs

This study was set up to establish the prevalence of Listeria monocytogenes in the tonsils of sows and fattening pigs from five Finnish slaughterhouses and to evaluate the genetic similarity of L. monocytogenes strains isolated from the tonsils. A total of 271 pig tonsils (132 tonsils from fattening pigs and 139 from sows) from five different slaughterhouses in various parts of Finland were studied from June 1999 to March 2000. Overall, 14 and 4% of pig tonsils harbored L. monocytogenes and Listeria innocua, respectively. The prevalence of L. monocytogenes in tonsils of fattening pigs (22%) was significantly higher than in sows (6%). The isolates (n = 38) recovered from tonsils showed a wide genetic diversity by means of 24 different pulsed-field gel electrophoresis (PFGE) types presented by the strains. Moreover, in numerical analyses of restriction patterns, no association was found between the clustering of strains and the slaughterhouses, and strains showing a similar PFGE type were recovered from pigs of different slaughterhouses. The high prevalence of L. monocytogenes showing various PFGE types in the tonsils of pigs could indicate a potential source of contamination of pluck sets, carcasses, and the slaughterhouse environment and of subsequent processing steps.

Role of flhA and motA in growth of Listeria monocytogenes at low temperatures

While temperature-dependent induction of flagella is a well-characterized phenomenon in Listeria monocytogenes, the essentiality of increased flagellum production during growth at low temperatures remains unclear. To study this relationship, we compared the relative expression levels of two motility genes, flhA and motA, at 3°C, 25°C and 37°C in L. monocytogenes strain EGD-e by using qRT-PCR, and compared the growth curves, motility, and flagellation between the wild-type and flhA and motA deletion mutants. The relative expression levels of flhA and motA at 3°C were significantly higher than at 37°C (p<0.01). At 3°C, the level of flhA transcripts was also significantly higher than at 25°C (p<0.01). Growth curve analysis showed that at 3°C both the growth rates and maximum optical densities of ΔflhA and ΔmotA strains at 600 nm were significantly lower than those of the wild-type (p<0.001), while no significant differences were observed between the wild-type and the mutants at 37°C, and 25°C. Mutant strains ΔflhA and ΔmotA were nonmotile at all three temperatures. At 25°C, the number of flagellated cells of ΔmotA was notably reduced compared with the wild-type, whereas ΔflhA appeared nonflagellated at all temperatures. The results suggest that flhA and motA play a role in the cold tolerance of L. monocytogenes strain EGD-e, and that motile flagella may be needed for optimal cold stress response of L. monocytogenes.

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

ABSTRACT Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.

Roles of Four Putative DEAD-Box RNA Helicase Genes in Growth of Listeria monocytogenes EGD-e under Heat, pH, Osmotic, Ethanol, and Oxidative Stress Conditions

ABSTRACT To examine the role of the four putative DEAD-box RNA helicase genes of Listeria monocytogenes EGD-e in stress tolerance, the growth of the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains at 42.5�C, at pH 5.6 or pH 9.4, in 6% NaCl, in 3.5% ethanol, and in 5 mM H2O2 was studied. Restricted growth of the Δlmo0866 deletion mutant strain in 3.5% ethanol suggests that Lmo0866 contributes to ethanol stress tolerance of L. monocytogenes EGD-e. The Δlmo1450 mutant strain showed negligible growth at 42.5�C, at pH 9.4, and in 5 mM H2O2 and a lower maximum growth temperature than the wild-type EGD-e, suggesting that Lmo1450 is involved in the tolerance of L. monocytogenes EGD-e to heat, alkali, and oxidative stresses. The altered stress tolerance of the Δlmo0866 and Δlmo1450 deletion mutant strains did not correlate with changes in relative expression levels of lmo0866 and lmo1450 genes under corresponding stresses, suggesting that Lmo0866- and Lmo1450-dependent tolerance to heat, alkali, ethanol, or oxidative stress is not regulated at the transcriptional level. Growth of the Δlmo1246 and Δlmo1722 deletion mutant strains did not differ from that of the wild-type EGD-e under any of the conditions tested, suggesting that Lmo1246 and Lmo1722 have no roles in the growth of L. monocytogenes EGD-e under heat, pH, osmotic, ethanol, or oxidative stress. This study shows that the putative DEAD-box RNA helicase genes lmo0866 and lmo1450 play important roles in tolerance of L. monocytogenes EGD-e to ethanol, heat, alkali, and oxidative stresses.

Comparative Phenotypic and Genotypic Analysis of Swiss and Finnish Listeria monocytogenes Isolates with Respect to Benzalkonium Chloride Resistance

Reduced susceptibility of Listeria monocytogenes to benzalkonium chloride (BC), a quaternary ammonium compound widely used in food processing and hospital environments, is a growing public health and food safety concern. The minimal inhibitory concentration of BC on 392 L. monocytogenes strains from Switzerland (CH) and Finland (FIN) was determined. Within this strain collection, benzalkonium chloride resistance was observed in 12.3% (24/195) of Swiss and 10.6% (21/197) of Finnish strains. In both countries, the highest prevalence of BC-resistant strains (CH: 29.4%; FIN: 38.9%) was detected among serotype 1/2c strains. Based on PCR analysis, genes coding for the qacH efflux pump system were detected for most of the BC-resistant strains (CH: 62.5%; FIN: 52.4%). Some Swiss BC-resistant strains harbored genes coding for the bcrABC (16.7%) efflux pump system, while one Finnish BC-resistant strain harbored the emrE gene previously only described among BC-resistant L. monocytogenes strains from Canada. Interestingly, a subset of BC-resistant strains (CH: 5/24, 20.8%; FIN: 9/21, 42.8%) lacked genes for efflux pumps currently known to confer BC resistance in L. monocytogenes. BC resistance analysis in presence of reserpine showed that the resistance was completely or partially efflux pump dependent in 10 out of the 14 strains lacking the known BC resistance genes. Sequence types 155 and ST403 were over-representated among these strains suggesting that these strains might share similar but yet unknown mechanisms of BC resistance.

Listeria monocytogenes Serotypes 1/2c and 3c Possess inlH

To examine the serotype specificity of inlH, which encodes the virulence-associated surface protein InlH related to the intracellular survival of Listeria monocytogenes in mice, the presence of inlH in 337 L. monocytogenes strains, representing 11 different serotypes, was studied. A total of 106 strains representing 3 serotypes and 14 pulsed-field gel electrophoresis (PFGE) types were positive for inlH by polymerase chain reaction. inlH was present in all 99 serotype 1/2c and 3 serotype 3c strains. Moreover, 4 out of 129 (3%) serotype 1/2a strains carried inlH. All 106 strains representing serotypes 1/2b, 3a, 3b, 4a, 4b, 4c, 4d, and 7 and 125 out of 129 (97%) serotype 1/2a strains were inlH-negative. The coding sequences of the inlH genes of eight L. monocytogenes strains representing three serotypes and five PFGE types were identical. These results suggest that inlH is specifically present in serotype 1/2c, 3c, and a small fraction of 1/2a L. monocytogenes strains and exists as a single allele.