Species-Level Discrimination of Psychrotrophic Pathogenic and Spoilage Gram-Negative Raw Milk Isolates Using a Combined MALDI-TOF MS Proteomics-Bioinformatics-based Approach.

Identification of psychrotrophic pathogenic and spoilage Gram-negative bacteria using rapid and reliable techniques is important in commercial milk processing, as these bacteria can produce heat-resistant proteases and act as postprocessing contaminants in pasteurized milk. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a proven technology for identification of bacteria in food, however, may require optimization for identification of pathogenic and spoilage bacteria in milk and dairy products. The current study evaluated the effects of various culture conditions and sample preparation methods on assigning of raw milk isolates to the species level by MALDI-TOF MS. The results indicated that culture media, incubation conditions (temperature and time), and sample preparation significantly affected the identification rates of bacteria to the species level. Nevertheless, the development of spectral libraries of isolates grown on different media using a web tool for hierarchical clustering of peptide mass spectra (SPECLUST) followed by a ribosomal protein based bioinformatics approach significantly enhanced the assigning of bacteria, with at least one unique candidate biomarker peak identified for each species. Phyloproteomic relationships based on spectral profiles were compared to phylogenetic analysis using 16S rRNA gene sequences and demonstrated similar clustering patterns with significant discriminatory power. Thus, with appropriate optimization, MALDI-TOF MS is a valuable tool for species-level discrimination of pathogenic and milk spoilage bacteria.

Microbiological quality of raw milk attributable to prolonged refrigeration conditions.

Refrigerated storage of raw milk is a prerequisite in dairy industry. However, temperature abused conditions in the farming and processing environments can significantly affect the microbiological quality of raw milk. Thus, the present study investigated the effect of different refrigeration conditions such as 2, 4, 6, 8, 10 and 12 °C on microbiological quality of raw milk from three different dairy farms with significantly different initial microbial counts. The bacterial counts (BC), protease activity (PA), proteolysis (PL) and microbial diversity in raw milk were determined during storage. The effect of combined heating (75 ± 0·5 °C for 15 s) and refrigeration on controlling those contaminating microorganisms was also investigated. Results of the present study indicated that all of the samples showed increasing BC, PA and PL as a function of temperature, time and initial BC with a significant increase in those criteria ≥6 °C. Similar trends in BC, PA and PL were observed during the extended storage of raw milk at 4 °C. Both PA and PL showed strong correlation with the psychrotrophic proteolytic count (PPrBC: at ≥4 °C) and thermoduric psychrotrophic count (TDPC: at ≥8 °C) compared to total plate count (TPC) and psychrotrophic bacterial count (PBC), that are often used as the industry standard. Significant increases in PA and PL were observed when PPrBC and TDPC reached 5 × 104 cfu/ml and 1 × 104 cfu/ml, and were defined as storage life for quality (S LQ), and storage life for safety (S LS) aspects, respectively. The storage conditions also significantly affected the microbial diversity, where Pseudomonas fluorescens and Bacillus cereus were found to be the most predominant isolates. However, deep cooling (2 °C) and combination of heating and refrigeration (≤4 °C) significantly extended the S LQ and S Ls of raw milk.

The effect of electromagnetic fields, from two commercially available water treatment devices, on bacterial culturability.

Commercially available pulsed-electromagnetic field (PEMF) devices are currently being marketed and employed to ostensibly manage biofouling. The reliable application and industry acceptance of such technologies require thorough scientific validation - and this is currently lacking. We have initiated proof-of-principle research in an effort to investigate whether such commercially available PEMF devices can influence the viability (culturability) of planktonic bacteria in an aqueous environment. Thus two different commercial PEMF devices were investigated via a static (i.e. non-flowing) treatment system. 'Healthy' Escherichia coli cells, as well as cultures that were physiologically compromised by silver nano-particles, were exposed to the PEMFs from both devices under controlled conditions. Although relatively minor, the observed effects were nevertheless statistically significant and consistent with the hypothesis that PEMF exposure under controlled conditions may result in a decrease in cellular viability and culturability. It has also been observed that under certain conditions bacterial growth is actually stimulated.

Comparison of identification systems for psychrotrophic bacteria isolated from raw bovine milk.

Psychrotrophic bacteria in raw milk produce heat-resistant extracellular proteases, resulting in spoilage and shelf-life reduction of ultrahigh temperature treated milk and milk products. Controlling of these spoilage microbes requires rapid and reliable identification systems for screening of raw milk. This study aimed to compare commercial bacterial identification systems with a genetic method (considered as the 'gold standard' method) for the identification of heat-resistant protease producing bacteria in raw milk. Five bacterial identification systems were compared based on typability, discrimination power (i.e. Simpson's Index of Diversity), reproducibility and speed of analysis. The accuracy of 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API, and Microbact for the identification of Gram negative bacilli at the species level was 100.0%, 86.8%, 63.2%, 60.5% and 57.9%, respectively. The Gram positive bacilli were identified by 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, and API with accuracies at the species level of 100.0%, 85.0%, 95.0% and 90.0%, respectively. The 16S rRNA gene sequencing and phylogenetic analysis discriminated Pseudomonas fluorescens, Pseudomonas syringae, Hafnia alvei, Bacillus cereus, Bacillus pumilus and Bacillus licheniformis to the subspecies level. The Simpson's Index of Diversity scores were 0.966, 0.711, 0.496, 0.472, and 0.140, for 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API and Microbact, respectively. Limited reference profiles in the databases of Biolog, MALDI-TOF MS, API and Microbact systems reduced their accuracy in bacterial identification, compared to 16S rRNA gene sequencing. The rapidity of each assay is in the following order; MALDI-TOF MS>16S rRNA gene sequencing>Biolog>Microbact>API. The reproducibility of the assays is in the order of 16S rRNA gene sequencing>API>Microbact>MALDI-TOF MS>Biolog. Thus, 16S rRNA gene sequencing appears to be the most reliable and robust system for the identification of dairy spoilage bacteria. The Biolog system is suitable for the identification of Gram negative spoilage bacteria, while MALDI-TOF MS and API systems are suitable for the identification of Gram positive spoilage bacteria isolated from raw milk. The commercial systems used in this study have been developed and extensively used for the identification of clinical microbes but only a limited number of studies used those systems to identify the environmental microorganisms that often contaminate raw milk. Therefore, comparison of those systems for the identification of spoilage microbes in raw milk would provide better understanding of their suitability for routine dairy microbiology and more extensive dairy research.

Telomerase activity in pleural malignant mesotheliomas.

New treatments are needed for malignant pleural mesothelioma (MPM), which currently has a poor prognosis. Cellular immortalisation, one of the hallmarks of cancer, depends on the activity of a telomere length maintenance mechanism (TMM) - either telomerase or alternative lengthening of telomeres (ALT). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. The aim of this study was to determine what proportion of MPMs use ALT and/or telomerase. Forty-three MPMs from 42 patients were examined for telomerase and ALT activity. Telomerase activity was detected by immunoaffinity purification followed by the telomere repeat amplification protocol (TRAP), and ALT activity was determined by the C-circle assay and by assessing telomere lengths using terminal restriction fragment analyses. We found that 43 of 43 MPMs were telomerase-positive[+] and ALT-negative[-]. Therefore, to investigate whether pleural mesothelial cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalisation, in which normal pleural mesothelial cells were transduced with simian virus 40 (SV40) oncogenes. We found that normal mesothelial cells were TMM-negative, and that expression of the SV40 oncogenes did not directly activate telomerase or ALT. Immortalisation, which in this experimental system results from additional genetic changes that have not yet been identified, was accompanied by activation of either TMM. Therefore, pleural mesothelial cells are capable of activating either TMM in vitro, and the observation that 100% of MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumour type. We conclude that MPM is a tumour that could be considered for anti-telomerase therapy.

DNA damage induces alternative lengthening of telomeres (ALT) associated promyelocytic leukemia bodies that preferentially associate with linear telomeric DNA.

The linear chromosomes of vertebrates terminate in telomeres that consist of a tandemly repeated hexameric sequence, 5'TTAGGG3'. Telomeres form a protective loop structure (t-loop), which is thought to prevent them from being recognized as a double-strand break. Approximately 10% of human tumors prevent shortening of their telomeres by using a recombination-mediated alternative lengthening of telomeres (ALT) mechanism. ALT-positive human cells contain extrachromosomal telomere repeat (ECTR) DNA that may either be circular or linear. It has been proposed that ECTR may be generated by recombination events involving the t-loop. A proportion of the cells within ALT-positive cell populations contain promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins that are called ALT-associated PML bodies (APB). Although the presence of APBs is very useful for determining whether tumors and cell lines use the ALT mechanism, the function of APBs is unknown. It has previously been shown that telomeric DNA is particularly susceptible to damage by hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine. We report here that these DNA-damaging agents induce both linear and circular ECTR DNA in ALT cells and increase the proportion of cells that contain APBs. We partially purified APBs and showed that the telomeric repeat DNA they contain is predominantly linear. We propose that a function of APBs is to sequester linear telomeric DNA.

A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.

PURPOSE AND EXPERIMENTAL DESIGN: Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative. RESULTS: To assay for ALT, we detected ALT-associated promyelocytic leukemia (PML) bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization. APBs are PML nuclear domains containing telomeric DNA and are a known hallmark of ALT in cell lines. The APB assay concurred with the standard ALT assay in 62 of 62 tumors and showed that 35% of 101 soft tissue sarcomas (STS), 47% of 58 osteosarcomas (especially younger patients), 34% of 50 astrocytomas, and 0% of 17 papillary thyroid carcinomas were ALT positive (ALT+). The prevalence of ALT varied greatly among different STS subtypes: malignant fibrous histiocytomas, 77%; leiomyosarcomas, 62%; liposarcomas, 33%; synovial sarcomas, 9%; and rhabdomyosarcomas, 6%. ALT correlated with survival in glioblastoma multiforme and occurred more often in lower-grade astrocytomas, but ALT+ and ALT- sarcomas were equally aggressive in terms of grade and clinical outcome. CONCLUSION: The APB assay for ALT is suitable for paraffin-embedded tumors. It showed that a substantial proportion of STS, osteosarcomas, and astrocytomas, but not papillary thyroid carcinomas use ALT. APB positivity correlated strongly with survival of patients with astrocytomas.

Alternative lengthening of telomeres in mammalian cells.

Some immortalized mammalian cell lines and tumors maintain or increase the overall length of their telomeres in the absence of telomerase activity by one or more mechanisms referred to as alternative lengthening of telomeres (ALT). Characteristics of human ALT cells include great heterogeneity of telomere size (ranging from undetectable to abnormally long) within individual cells, and ALT-associated PML bodies (APBs) that contain extrachromosomal telomeric DNA, telomere-specific binding proteins, and proteins involved in DNA recombination and replication. Activation of ALT during immortalization involves recessive mutations in genes that are as yet unidentified. Repressors of ALT activity are present in normal cells and some telomerase-positive cells. Telomere length dynamics in ALT cells suggest a recombinational mechanism. Inter-telomeric copying occurs, consistent with a mechanism in which single-stranded DNA at one telomere terminus invades another telomere and uses it as a copy template resulting in net increase in telomeric sequence. It is possible that t-loops, linear and/or circular extrachromosomal telomeric DNA, and the proteins found in APBs, may be involved in the mechanism. ALT and telomerase activity can co-exist within cultured cells, and within tumors. The existence of ALT adds some complexity to proposed uses of telomere-related parameters in cancer diagnosis and prognosis, and poses challenges for the design of anticancer therapeutics designed to inhibit telomere maintenance.