Polymorphic variability in the exon 19 of the RB1 gene and its flanking intronic sequences in HPV16-associated precancerous lesions in the Greek population.

PURPOSE: The tumour suppressor protein RB plays a decisive role in negative control of the cell cycle, inhibiting tumour development. The present analysis investigated the prevalence of the nucleotide polymorphism A153104G, which is located at intron 18 of the RB1 gene, and investigated the impact of the polymorphic variability in the exon 19 and its flanking intronic sequences on the severity of cervical disease in HPV16-positive Greek women. METHODOLOGY: The nucleotide polymorphism A153104G was detected by PCR-RFLP assay, while the amplicons were further subjected to cloning and sequencing. Moreover, molecular evolutionary analysis was performed using the maximum-likelihood (ML) and empirical Bayesian (EB) methods in order to evaluate the selective pressure acting on exon 19 of the RB1 gene.Results/Key findings. The A153104G nucleotide polymorphism was only detected in one control case. Moreover, sequence analysis of the amplicons revealed that the polymorphic variability in the RB1 gene increased with the severity of the cervical dysplasia. The link between the observed polymorphic variability and the progress of cervical disease was reflected in the molecular evolutionary analysis that was performed on the exon 19 of the RB1 gene, since negative selective pressure was acting upon exon 19 in the control and low-grade squamous intraepithelial lesion (LSIL) cervical samples, while positive selective pressure was acting upon exon 19 in the high-grade squamous intraepithelial lesion (HSIL) specimens. CONCLUSIONS: The A153104G nucleotide polymorphism did not emerge as a potential biomarker for the development of precancerous lesions in the Greek patients, while the accumulation of sequence variations in RB1 gene might influence patients' susceptibility towards the progression of cervical neoplasia.

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10-2 CCID50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 105 and 1 CCID50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 105 CCID50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses.

Transfer of verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 from fleece to carcass during sheep slaughter in an Irish export abattoir.

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from fleece to dressed carcasses of 500 sheep, and to establish the virulence potential of recovered VTEC. Individual sheep were tracked and sampled (10 g fleece, full carcass swab) through the slaughter process. Samples were examined for the presence of verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay and positive samples were further screened for the presence of the above five serogroups by real-time PCR. VTEC cells were recovered from PCR positive samples by serogroup specific immunomagnetic separation and confirmed by serogroup specific latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR and isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). VTEC O26 was recovered from 5/500 (1.0%) fleece and 2/500 (0.4%) carcass samples. VTEC O157 was isolated from 4/500 (0.8%) fleece samples and 3/500 (0.6%) carcass samples. E. coli O103 was recovered from 84/500 (16.8%) fleece and 68/500 (13.6%) carcasses, but only one E. coli O103 isolate (0.2%) carried vt genes. E. coli O145 was recovered from one fleece sample, but did not carry vt genes. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from fleece to carcass was not observed with PFGE showing that VTEC O26 isolates from a matched fleece/carcass "pair" were not identical. This study shows that while VTEC O157 are being carried by sheep presented for slaughter in Ireland, other potentially clinically significant verotoxin producing strains (particularly VTEC O26) are emerging.

Characterization of a potentially novel 'blown pack' spoilage bacterium isolated from bovine hide.

AIMS: To characterize a psychrotrophic bacterium, designated TC1, previously isolated from a cattle hide in Ireland, and to investigate the ability of this strain to cause 'blown pack' spoilage (BPS) of vacuum-packaged beef primals. METHODS AND RESULTS: TC1 was characterized using a combination of phenotypic, chemotaxonomic and genotypic analyses and was assessed for its ability to spoil vacuum-packaged beef at refrigerated temperatures. TC1 was Gram-positive and formed elliptical subterminal endospores. The strain was able to grow between 0 and 33 °C, with optimal growth between 23 and 24 °C. TC1 could be differentiated from its phylogenetically closest neighbour (Clostridium lituseburense DSM 797(T)) by 16S rRNA gene sequencing, pulsed-field gel electrophoresis and cellular fatty acid composition. TC1 spoiled (BPS) beef within 42 days when inoculated in cold-stored (1 °C) vacuum-packed beef. CONCLUSIONS: The phenotypic, chemotaxonomic and genotypic characterization indicated that TC1 may represent a potentially novel, cold-tolerant, gas-producing bacterium of considerable economic significance to the beef industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports and characterizes an emerging BPS bacterium, which should be considered in future activities designed to minimize the psychrophilic and psychrotrophic spoilage of vacuum-packaged beef.

Diversity of culturable psychrophilic and psychrotrophic anaerobic bacteria isolated from beef abattoirs and their environments.

This study identified 431 psychrophilic or psychrotrophic isolates from commercial Irish beef abattoir environments and "blown packs" of vacuum-packed beef, using PCR and 16S rRNA sequencing, and estimated their intraspecies genetic diversity using restriction fragment length polymorphism (RFLP) analysis and spacer region PCR (SR-PCR). Twenty-five species were identified in the 431 isolates, with the most frequently recovered species being Clostridium gasigenes (n=315), Clostridium estertheticum (n=17), and a potentially novel species designated strain TC1 (n=52). These species were previously found to be associated with a particular type of spoilage known as blown-pack spoilage (BPS), which occurs in chilled-stored (i.e., -1.5°C to 4°C) vacuum-packaged meat within 2 to 4 weeks and involves the production of large volumes of gas. Overall, the study demonstrates the considerable and not previously reported diversity of the anaerobic microflora in abattoirs and the presence of a wide range of organisms capable of causing BPS at chilled temperatures.

The effect of storage temperature and inoculum level on the time of onset of 'blown pack' spoilage.

AIMS: To examine the effect of storage temperature and inoculum level on the time of onset of 'blown pack' spoilage (BPS) caused by psychrotolerant bacteria in vacuum-packed (VP) meats. METHODS AND RESULTS: Gas-producing species and strains (n = 11), recovered in our laboratory or reported as associated with BPS, were inoculated onto beef or lamb meat pieces at final levels of <10, 10, 10(2) and 10(3) CFU cm(-2), VP and stored at -1.5, 1 or 4 degrees C. Six strains produced observable amounts of gas within 42 days and a further four strains produced gas within 100 days. BPS was observed earliest in VP meats inoculated with Clostridium estertheticum ssp. estertheticum at all inoculum levels/storage temperature combinations examined. Storage temperature and inoculum level significantly affected (P < 0.001 and P < 0.05 respectively) the onset of BPS in all cases. CONCLUSIONS: Controlling contamination levels and lowering the storage temperature delay the onset of BPS. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the positive effects of low contamination-low temperature as control interventions preventing/delaying BPS in VP chilled meats and identifies some of the contaminants most likely to cause BPS in chilled stored VP meat products.

Isolation and sources of 'blown pack' spoilage clostridia in beef abattoirs.

AIMS: (i) To evaluate methods for isolation and molecular detection of blown pack spoilage (BPS) clostridia and (ii) to survey beef abattoirs for sources and distributions of Clostridium estertheticum and Cl. gasigenes. METHODS AND RESULTS: Molecular detection and conventional isolation methods were used to detect and recover BPS associated clostridia (Cl. estertheticum and Cl. gasigenes), from four commercial Irish beef abattoirs and their environments, during a one year study. DNA-based methods detected 218 Cl. estertheticum and 300 Cl. gasigenes, from 1680 samples, whereas culture-methods only yielded 17 Cl. estertheticum and 176 Cl. gasigenes isolates. BPS Clostridia were frequently detected in beef abattoirs and their environments, especially at areas prior to hide removal. The study noted a higher percentage of positive samples during the month of May (38.6%). CONCLUSIONS: (i) DNA-based techniques are the most reliable ways to determine the presence of these organisms in various samples and (ii) hides and faeces are the main reservoirs of BPS clostridia in the abattoirs. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides useful information to detect BPS organisms, as well as to develop a science-based control strategy of the problem.

Characterization of the microbial flora from a traditional Greek fermented sausage.

The microbial flora of naturally fermented sausages was studied. Lactic acid bacteria were the dominant species at the end of fermentation in all 3 batches (ca. 10(8) cfu g(-1)). Enterobacteria, Pseudomonas, yeasts and aerobic spore-formers decreased during fermentation and the ripening process and were below the detection limit in the end product. Enterococci exceeded 10(4)-10(5) cfu g(-1) during fermentation and remained constant at this level during ripening. Gram-positive, catalase-positive cocci exceeded 10(5) cfu g(-1), except for batch 1, during the first days of fermentation and then decreased until the end of ripening (10(2)-10(4) cfu g(-1)). No pathogenic staphylococci, sulfite reducing clostridia or Salmonella spp. were detected. Listeria spp. occurred in the first days of fermentation but were eliminated by the end of whole process in all batches. Identification showed that the majority of lactobacilli isolated from MRS agar strains were assigned to the species of Lactobacillus plantarum and Lb. plantarum/pentosus. All the isolated strains from the mannitol salt agar belonged to the genus of Staphylococcus. The predominant species were Staphylococcus saprophyticus, Staphylococcus xylosus and Staphylococcus simulans. The tests used to characterize the lactic acid bacteria and staphylococci as well as their distribution on the three batches were also discussed.