Identification of stringent response-related and potential serological proteins released from Bacillus anthracis overexpressing the RelA/SpoT homolog, Rsh Bant.

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.

Aerobic plate counts as a measure of hazard analysis critical control point effectiveness in a pork processing plant.

A hazard analysis critical control point (HACCP) system was designed to identify specific hazards so that preventive and control measures to ensure the safety of a food could be implemented. Microbiological data generated through sampling were used to characterize the hygienic performance and to validate and verify the various HACCP plans. Aerobic plate counts (APCs) often are chosen as an indicator of the effectiveness of HACCP plans, because data for all aerobic bacteria are more easily collected than are data for pathogens of concern or other indicator organisms. However, it is not clear whether APCs are useful in verifying that a HACCP plan is working satisfactorily. In this study, APC data were collected from one pork-cutting plant in Korea both before and after the company initiated its HACCP plan. These APC data were used to compare microbiological differences and to determine the effect of any changes before and after implementing the HACCP plan. For this pork plant, after the HACCP plan was implemented the proportion of samples exceeding the 3 log CFU/cm2 limit dropped from 73.39 to 4.29% for the overall process. These results indicate that this plant improved its hygienic performance considerably and that the HACCP plan was an effective and valuable tool for achieving this improvement. The APC data were sufficient for validation and verification of the HACCP system that was successfully implemented to improve hygienic performance.

Modeling the level of contamination of Staphylococcus aureus in ready-to-eat kimbab in Korea.

The risk of Staphylococcus aureus in ready-to-eat kimbab (rice rolled in laver) sold in Korea was evaluated by a mathematical modeling approach. Four nodes were constructed from preparation at retail to consumption. A predictive microbial growth model and survey data were combined with probabilistic modeling to simulate the level of S. aureus in a single kimbab at the time of consumption. We estimated the mean level of S. aureus to be 2.92 log CFU/g for a typical kimbab (150 to 200 g each) at the time of consumption. Our model also showed that 29.73% of the kimbabs had > or = 100,000 S. aureus CFU/g, which poses some risk of illness, since some level of enterotoxin would be expected from toxigenic strains. However, because of the lack of dose-response models for staphylococcal enterotoxin, the final level of S. aureus in the kimbabs could not be used to estimate how many people would become ill from eating them. Correlation sensitivity results showed that consumer eating patterns and initial contamination levels at retail stores were the most significant risk factors for illness and that temperature control under 10 degrees C was a critical control point in kimbab retail establishments to prevent the growth of S. aureus.

Comparison of four molecular typing methods for the differentiation of Salmonella spp.

A total of 57 strains of Salmonella spp. were differentiated by random amplification of polymorphic DNA (RAPD) fingerprinting using three different primers (OPL-03, primer 1, and primer A); by Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting; by ribotyping-PCR; and by Single Strand Conformation Polymorphism (SSCP). From the 57 strains, RAPD fingerprinting with primers OPL-03, 1, and A produced 42, 51, and 54 fingerprint patterns respectively. ERIC fingerprinting produced 50 patterns; ribotyping-PCR produced four patterns, and SSCP produced 11 patterns. Combinations of two different typing methods generally increased the discrimination of Salmonella strains. A combination of two different RAPDs or a combination of RAPD and ERIC was better than the other combinations. Discrimination using a combination of RAPD (primer 1 or primer A) and ERIC, which could differentiate all 57 Salmonella strains, was better than the combination of two RAPDs. This study indicated that the use of a combination of RAPD (primer 1 or primer A) and ERIC should be useful for the differentiation of field-isolated Salmonella strains and epidemiological studies.

Rapid enumeration of Listeria monocytogenes in milk using competitive PCR.

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk. Sterile milk was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR. Several primers for L. monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected. The primer set produced a 636-bp band from L. monocytogenes, but no band appeared from the other six Listeria spp. tested. A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set. When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR. For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA. The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site. Known amount of competitor DNA was coamplified with L. monocytogenes total DNA isolated from artificially inoculated milk. The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR. The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method. For the whole procedure, it took only 5 h.