Effects of different media on the enrichment of low numbers of Shiga toxin-producing Escherichia coli in mung bean sprouts and on the development of the sprout microbiome.

Sprouted seeds have been implicated in a number of serious outbreaks caused by Salmonella and Shiga toxin-producing Escherichia coli. Sprouts pose a very complex challenge to bacterial pathogen enrichment and detection since they naturally contain high levels of background microflora including members of the Enterobacteriaceae. As such, the currently used method cannot ensure reliable detection of STEC in sprouts. In this study, we compared different media for the enrichment of Enterobacteriaceae in their ability to promote the growth of stressed STEC at 37°C and 42°C. Mung bean sprouts were spiked with low levels of STEC and their growth was recorded over time. In addition, the microbiome of mung bean sprouts was analysed before and after enrichment. Our results indicate that the growth of dry-stressed STEC is comparable in all of the tested enrichment media except for mTSB+Novobiocin and not influenced by the incubation temperature. Low levels of STEC spiked into the sprouts resuspended in media only grew to levels of around 4logcfu/ml during enrichment, which could reduce the probability of detection. Proteobacteria was the dominant phylum detected within the microbiome of non-enriched mung bean sprouts. During enrichment in EE-broth, Proteobacteria remained the most abundant phylum. In contrast, during enrichment in BPW the relative abundance of Proteobacteria decreased whereas Firmicutes increased when compared to the non-enriched mung bean sprout microbiome. The microbiome composition was not significantly influenced by the incubation temperature during enrichment in both BPW and EE-broth. This is the first study to examine the microbiome on sprouted mung bean seeds during BPW and EE enrichment and relates the bacterial community composition changes to the enrichment of pathogens.

Function impairing mutations in blaZ and blaR genes of penicillin susceptible Staphylococcus aureus strains isolated from bovine mastitis.

Molecular based approaches have gained increasing importance in routine mastitis diagnostics for typing and antibiotic resistance testing of Staphylococcus aureus. Out of 78 S. aureus strains isolated from bovine mastitis milk 10 of them harbored blaZ, blaI and blaR genes. Although 5 strains were phenotypically resistant to penicillin, the other 5 (all belonging the clonal complex 8) were penicillin susceptible. PCR amplification confirmed the presence of the blaZ, blaR and blaI genes in all 5 strains. Sequencing of these genes uncovered a 29 base deletion within the blaZ gene in all these strains that causes a translational frame shift, which is predicted to induce abrogation of BlaZ expression. Additionally single nucleotide insertions and deletions were detected in blaR of 3 strains. These insertions cause translation reading frame shifts and premature stop codons that are predicted to induce expression of truncated BlaR proteins. Using the genetically altered blaZ genes detected as targets, a real-time PCR system for detecting CC8 associated blaZ positive S. aureus strains that still remain susceptible to penicillin was developed. Such strains are part of detection challenges that must be considered in routine application of genotypic resistance testing of bovine mastitis S. aureus.

Les techniques moléculaires pour typiser les S. aureus et pour tester ces souches quant à la présence des gènes de résistance aux antibiotiques gagnent régulièrement en importance dans le diagnostic des mammites. Sur 78 souches de S. aureus, isolées de mammites bovines, 10 présentaient les gènes blaZ, blaI et blaR. Toutefois seules 5 de ces souches présentaient une résistance à la pénicilline, les 5 autres (appartenant toutes au complexe clonal 8) étaient sensibles à la pénicilline. On a pu confirmer par PCR la présence des gènes blaI et blaR dans ces 5 dernières souches. Le séquençage du gène blaZ a montré dans toutes ces souches une délétion importante de 29 bp avec pour conséquence que la protéine BlaZ n'était pas exprimée. En outre on a trouvé sur les gènes blaR de 3 souches des insertions et des délétions de nucléotides isolés. Cela conduit à des stop codons prématurés et donc à l'expression de protéines BlaR modifiées. En se basant sur les gènes blaZ modifiés, on a établi une PCR en temps réel pour identifier les souches de S. aureus du complexe clonal 8 sensibles à la pénicilline. De telles souches représentaient jusqu'à maintenant un défi dans l'utilisation de routine de la PCR dans le diagnostic des mammites.

Evaluation of cold growth and related gene transcription responses associated with Listeria monocytogenes strains of different origins.

The cold growth phenotypes and transcriptional activation of cold stress adaptation genes was evaluated amongst Listeria monocytogenes strains from human listeriosis cases, food products and associated production environments. Significant cold growth phenotypic variation was observed during growth of such strains in rich (BHI) as well as chemically defined minimal (MDM) nutrient conditions. While all twenty analyzed strains grew in BHI at 4 degrees C, only eight of these strains, mostly those recovered from human listeriosis cases, were also able to grow in MDM under similar cold stress. The cold growth phenotypes observed in BHI were used to define two categories of five strains each, which either displayed enhanced and poor cold tolerance relative to the rest of the strain collection. The first group (GP1) consisted of strains characterized by short lag times, whilst the second group (GP2) comprised of strains displaying prolonged lag times before growth resumption during incubation in BHI cultures at 4 degrees C. Transcription level activation of sigB, cspA and pgpH gene expression associated with cold stress exposure in a selection of GP1 and GP2 strains was assessed. Despite similar cold dependent sigB transcript induction between these two strain groups, there were significant differences observed in cold stress dependent induction of cspA and pgpH transcripts. Cold tolerant GP1 strains displayed relatively higher transcriptional activation of cspA and pgpH after cold stress exposure compared to the cold sensitive GP2 strains. This study highlights strain variability in cold stress tolerance phenotypes, as well as in strain capacity to activate specific cold adaptation gene expression responses. In addition the study also shows that enhanced and poor cold growth phenotypes are associated with particular strain capacity to activate important cold stress gene expression responses upon transition of L. monocytogenes into low temperature environments.

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level.

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.

Growth characteristics of Listeria monocytogenes, Listeria welshimeri and Listeria innocua strains in broth cultures and a sliced bologna-type product at 4 and 7 degrees C.

The growth characteristics of meat processing plant-derived field strains of Listeria monocytogenes, L. welshimeri and L. innocua were analyzed. The strains were inoculated in BHI broth cultures and incubated at 4 and 7 degrees C. Growth curves were determined by colony counting for 28 days. Significant variations were detected in the growth properties of these field-derived strains. In particular some of the L. monocytogenes strains displayed better cold stress tolerance. These discrepancies in growth behavior were more apparent in the cultures at 4 degrees C compared to 7 degrees C. Similar growth characteristics were observed for selected L. monocytogenes strains also in food challenge tests based on a sliced bologna-type product. The results stress the need for more evaluation of field strain growth characteristics and incorporation of such information in relevant predictive microbial growth models for L. monocytogenes risk assessment in naturally contaminated food products.

Application of an F57 sequence-based real-time PCR assay for Mycobacterium paratuberculosis detection in bulk tank raw milk and slaughtered healthy dairy cows.

A light cycler-based real-time PCR assay that targets the F57 sequence was used to collect data on the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in 100 bulk tank raw milk samples and in a population of 101 slaughtered dairy cattle. The assay's reproducible detection limit in total genomic DNA templates isolated from 10-ml samples of MAP-spiked raw milk was 100 cells per ml. Similarly, the evaluation of MAP-spiked bovine feces also demonstrated that the assay had a reproducible detection limit of 100 cells if they were contained within 200 mg of fecal sample material. Among the 100 bulk tank milk samples that were tested, we found 3 samples (3%) to be positive for MAP In the slaughterhouse part of the study, 8.9% (9 of 101) of the cows were positive for MAP DNA in fecal samples, 4.9% (5 of 101) in mesenteric lymph nodes, 0.9% (1 of 101) in ileum tissue, and 3.6% (3 of 84) in milk. Meanwhile, for 2.9% (3 of 101) of the culled cows, MAP DNA was detected in samples of diaphragmatic muscles.

Cold stress tolerance of Listeria monocytogenes: A review of molecular adaptive mechanisms and food safety implications.

The foodborne pathogen Listeria monocytogenes has many physiological adaptations that enable survival under a wide range of environmental conditions. The microbes overcome various types of stress, including the cold stress associated with low temperatures in food-production and storage environments. Cold stress adaptation mechanisms are therefore an important attribute of L. monocytogenes, enabling these food pathogens to survive and proliferate to reach minimal infectious levels on refrigerated foods. This phenomenon is a function of many molecular adaptation mechanisms. Therefore, an improved understanding of how cold stress is sensed and adaptation measures implemented by L. monocytogenes may facilitate the development of better ways of controlling these pathogens in food and related environments. Research over the past few years has highlighted some of the molecular aspects of cellular mechanisms behind cold stress adaptation in L. monocytogenes. This review provides an overview of the molecular and physiological constraints of cold stress and discusses the various cellular cold stress response mechanisms in L. monocytogenes, as well as their implications for food safety.

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay.

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

Relevant aspects of Arcobacter spp. as potential foodborne pathogen.

Arcobacter species are Gram-negative spiral-shaped organisms belonging to the family Campylobacteraceae that can grow microaerobically or aerobically. The Arcobacter organisms also have the ability to grow at 15 degrees C, which is a distinctive feature that differentiates Arcobacter species from Campylobacter species. Cultural detection of Arcobacter is generally performed by an enrichment step and takes 4 to 5 days. In the last few years, several studies comparing different culture-based protocols have been published. Furthermore, DNA-based assays have also been established for rapid and specific identification of Arcobacter spp. Recent evidence suggests that Arcobacter, especially A. Butzleri, may be involved in human enteric diseases. Moreover, A. butzleri has also occasionally been found in cases of human extraintestinal diseases. However, up to now, little is known about the mechanisms of pathogenicity or potential virulence factors of Arcobacter spp. There is evidence that livestock animals may be a significant reservoir of Arcobacter spp. and over the last few years, the presence of these organisms in raw meat products as well as in surface and ground water has received increasing attention. In view of control measures to be used to prevent or to eliminate the hazard of Arcobacter spp. in food, several treatments have been evaluated for their effectiveness. While the role of Arcobacter spp. in human disease awaits further evaluation, a precautionary approach is advisable. Measures aimed at reduction or eradication of Arcobacter from the human food chain should be encouraged. With this article, we review the recent literature on this organism with a special emphasis on the information relevant to food safety.

Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle.

A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits. The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains. Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic-uremic syndrome (HUS). Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes. Of strains encoding for stx2 variants, 63% were positive for stx2 subtype. Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively. Amongst the seven intimin-positive strains, one possessed intimin type beta1 (O5:H-), one intimin gamma1 (O145:H), one intimin gamma2/theta, (O111:H21), and four intimin epsilon (O103:H2). The strains belonged to 29 serovirotypes (association between serotypes and virulence factors). O103:H2 stx1eae-epsilon ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains. Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-gamma1ehxA showed a pattern of highly virulent human strains. This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans.

HIV-1 reverse transcriptase and integrase enzymes physically interact and inhibit each other.

Ordered molecular interactions and structural changes must take place within the human immunodeficiency virus type 1 (HIV-1) preintegration complex at various stages for successful viral replication. We demonstrate both physical and biochemical interactions between HIV-1 reverse transcriptase and integrase enzymes. This interaction may have implications on the in vivo functions of the two enzymes within the HIV-1 replication complex. It may be one of the various molecular interactions, which facilitate efficient HIV-1 replication within the target cells.

Functional genomics in HIV-1 virus replication: protein-protein interactions as a basis for recruiting the host cell machinery for viral propagation.

Identification and characterization of protein-protein interactions between the host cell and parasites both enhance our understanding of basic cell biology and provide insights into central processes of parasite life cycles. Research on HIV-1 has broadened our knowledge of the various molecular events involved. However, our understanding of how this virus interacts with the host cell at the level of protein-protein interaction is still limited. Through these interactions the virus is able to recruit certain cellular metabolic pathways for its replication. Here we summarize our current knowledge of protein-protein interactions between HIV-1 and host cell factors during viral replication.

Intramolecular chimeras of the p51 subunit between HIV-1 and FIV reverse transcriptases suggest a stabilizing function for the p66 subunit in the heterodimeric enzyme.

The human immunodeficiency virus (HIV) reverse transcriptase (RT) is a heterodimeric enzyme composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. Recently we showed that p51 plays an important role in the conformation of p66 within the HIV-1 RT heterodimer and hence appears to influence its catalytic activities [Amacker, M., and H ubscher, U. (1998) J. Mol. Biol. 278, 757-765]. This was further investigated here via construction of three intramolecular chimeras of HIV-1 and FIV RTs. The first 25 and 112 amino acids of the N terminus, respectively, as well as the last 22 amino acids of the C terminus in the p51 subunit of HIV-1 RT were exchanged with the corresponding regions of the FIV RT and combined with the wild-type HIV-1 p66. Characterization of these chimeric RT heterodimers demonstrated significant biochemical differences in (i) DNA-dependent DNA synthesis, (ii) strand displacement DNA synthesis, and (iii) RNase H activity. Our results indicate that both the N and C termini of HIV-1 RT p51 appear to be important in stabilizing the RT heterodimer for enzymatic functions.