Robert E. Levin

Incidence and enumeration of Vibrio parahaemolyticus in shellfish from two retail sources and the genetic diversity of isolates as determined by RAPD-PCR analysis

Robert E. Levin et al. — Thu, 19 Jan 2012 10:35:15 PST

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.

Concentration and tracking of Listeria monocytogenes strains in a seafood-processing environment using a most-probable number enrichment procedure and randomly amplified polymorphic DNA analysis

Robert E. Levin et al. — Thu, 19 Jan 2012 10:34:22 PST

Concentrations of environmental microflora and Listeria monocytogenes were monitored at multiple environmental locations within a seafood-processing facility over the course of 6 months. Concentrations of L. monocytogenes were determined using a most-probable-number (MPN) enrichment procedure. Two floor drains had persistent low concentrations of L. monocytogenes (0.03 to >1,100 MPN/cm2). In comparison, concentrations of the other organisms in the drain were much higher (heterotrophic plate count range of 10(5) to 10(8) CFU/cm2). Concentrations of environmental organisms (heterotrophic aerobic plate counts and counts of pseudomonads, Shewanella spp., Aeromonas hydrophila, and coliforms) were not correlated with concentrations of L. monocytogenes. The 178 confirmed L. monocytogenes isolates from the MPN procedure were further characterized by randomly amplified polymorphic DNA analysis. Sixteen different banding patterns were identified, and nine of the patterns were identified from samples collected on two or more collection dates. From all locations, banding type A was observed in 98 confirmed isolates (55%). Although present, L. monocytogenes was a relatively minor component in the ecosystem of the floor drains in this seafood-processing facility.

Quantitative detection of Vibrio vulnificus by competitive polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:32:44 PST

Quantitative detection of V. vulnificus in shellfish via competitive PCR was herein developed. We designed a forward primer, a reverse primer and a hybrid forward primer based on the V. vulnificus specific hemolysin gene (vvh), which was used to amplify target DNA and an internal competitive PCR standard. The specificity of the primers for V. vulnificus was proven by gel electrophoresis. The detection sensitivity for V. vulnificus was 220 CFU per PCR reaction with cells from a pure culture and 270 CFU/g of tissue without enrichment. After 10 hours of enrichment at 37° C the minimum detection level was reduced to 7 CFU/g of tissue.

Effects of primers and Taq polymerase on randomly amplified polymorphic DNA analysis for typing Listeria monocytogenes from the environment of a shrimp processing plant

Robert E. Levin et al. — Thu, 19 Jan 2012 10:31:44 PST

Ninety-nine randomly selected isolates of Listeria monocytogenes obtained during a 5 month sampling period from several processing environment locations in a shrimp processing plant were subjected to RAPD analysis with the use of 4 primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. 18 composite RAPD types were discerned with the use of the 4 primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates and type 9 comprised 49 indistinguishable isolates. Results indicated that the shrimp processing plant was dominated by these 2 RAPD types, which comprised 63.6% of the 99 randomly selected isolates.

Detection, enumeration, and RAPD analysis of Listeria monocytogenes isolates in fish derived from retail sources in Western Massachusetts

Robert E. Levin et al. — Thu, 19 Jan 2012 10:29:40 PST

Fish were sampled over a 24-month period from two major supermarket retail outlets in Hadley, Massachusetts, USA, designated A and B, for the incidence of Listeria monocytogenes and numbers of the organism present per 100 g of tissue. Fifteen species of fish were represented. Seventy-four samples out of a total of 320 were confirmed by PCR as yielding L. monocytogenes. From retail source A, a total of 171 samples yielded 59 (34.5%) that were positive for the presence of L. monocytogenes. In contrast, from retail source B, a total of 149 samples yielded 15 (10.0%) that were positive. Only six samples (3.5%) from retail source A had MPN counts of L. monocytogenes in the range of 100 to 1,000 per 1,000 g. Only two samples (1.3%) from retail source B had counts of L. monocytogenes from 100 to 1,000 per 100 g. A total of 221 strains of L. monocytogenes were derived from the MPN cultures, 164 from retail source A, and 57 from retail source B. All 221 strains were subjected to RAPD analysis using three random primers. Primer LMPB1 yielded 21 RAPD profiles, primer LMPB4 yielded 19 profiles, and primer HLWL74 yielded 26 profiles. A total of 55 composite profiles were identified by combining the profiles derived from the three primers. Source A yielded 50 composite RAPD profiles, whereas source B yielded only ten composite profiles. In addition, 27 of the 55 composite profiles were derived from individual isolates and RAPD types 11 and 18 included 49 and 27 isolates respectively. Fish from retail source A clearly harbored far more RAPD types than did source B. The results clearly indicated that two major retail sources in close geographic proximity can vary considerably with respect to the incidence and numbers of L. monocytogenes present on the fish tissue. It was not possible to determine whether the processors furnishing fish to retail outlet A or the supermarket itself was responsible for the notably higher incidence and numbers of L. monocytogenes on fish from retail source A compared to fish from retail source B.

Vibrio vulnificus, a notably lethal human pathogen derived from seafood: a review of its pathogenicity, subspecies characterization, and molecular methods of detection

Robert E. Levin — Thu, 19 Jan 2012 10:27:33 PST

Vibrio vulnificus is presently considered the most infectious and lethal of all human pathogenic vibrios. The organism requires at least 0.5 NaCl for growth, is naturally ubiquitous to marine coastal waters and shellfish, and is sensitive to refrigeration temperatures. Septisemic infections by the organism usually result from the consumption of raw shellfish. Three biotypes are presently recognized and distinguished on the basis of biochemical characteristics, serology, and molecular typing. Cirrhosis of the liver due to chronic alcoholism is considered a high risk factor for infection by this organism, presumably due to increased levels of serum iron released by damaged hepatocytes. The organism produces an unusually large number of extracellular virulence factors. A number of selective agar media have been developed for isolation of the organism incorporating various levels of the antibiotics colistin, polymyxin-B, bile salts, and K tellurite. The molecular techniques applied to detecting, identifying, and characterizing the organism include: the polymerase chain reaction (PCR), real-time PCR, random amplified polymorphic DNA analysis (RAPD), multiplex PCR, ribotyping, multilocus enzyme electrophoresis (MLEEE), PCR-restriction fragment length polymorphism (PCR-RFLP) and gene sequence typing. In addition, seminested reverse transcription-PCR (RT-PCR) has been used to detect viable but non-culturable (VBNC) cells of V. vulnificus.

Quantification of Vibrio vulnificus using the polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:26:56 PST

An efficient procedure was developed for the quantitative PCR detection of Vibrio vulnificus in pure culture. The procedure involved boiling cell suspensions in TZ solution (2% Triton X-100 and 2.5 mg mL–1 NaN3 in 0.1 M Tris-HCl buffer at pH 8.0). Serial dilutions of lysed cell suspensions were then used for PCR. The method of visualizing amplified bands in agarose gels was evaluated by comparing a new nucleic acid dye (GelStar) to ethidium bromide (EB). GelStar stain was found to yield discernible amplified bands with lower levels of target DNA than could be achieved with EB. The minimum detection level with the GelStar stain was 16 CFU per PCR reaction compared to 40 CFU with EB. The relative fluorescence intensity of the DNA bands was analyzed with the NIH Image 1.61 software program. Calibration curves relating fluorescence intensity of amplified bands to lysed cells were obtained and the method was found suitable for the quantification of genomic DNA derived from 101–103 CFU per PCR reaction.

Quantitative determination of Vibrio parahaemolyticus by polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:26:05 PST

A method for quantitative detection of Vibrio parahaemolyticus, based on the polymerase chain reaction (PCR), was developed. Several lysis methods were compared and a recently developed lysis solution proved effective. The PCR amplification products were visualized after agarose gel electrophoresis with the nucleic acid stains GelStar and ethidium bromide. The relative fluorescent intensity of the DNA bands was analyzed using the NIH Image 1.61 software program. The GelStar stain was found to be more sensitive than ethidium bromide. The limit of detection by staining DNA bands with GelStar was 16 CFU per PCR and 48 with ethidium bromide. A log-linear relationship between the number of CFU per PCR reaction and the fluorescent intensity of the DNA bands was obtained and calibration curves were generated.

The application of real-time PCR to food and agricultural systems. A review

Robert E. Levin — Thu, 19 Jan 2012 10:25:10 PST

Real-time PCR (RT-PCR) allows each cycle of DNA amplification to be observed on a computer screen throughout the sequence of thermal cycling, hence the designation "real-time." RT-PCR assays have been developed for a variety of target sequences of food borne microbial pathogens, plant pathogens, and genetically modified foods. A major and highly sensitive aspect of RT-PCR is the use of fluorescent reporter dyes that undergo enhanced fluorescence when bound to DNA. Among the fluorescent reporter systems employed are SYBR green, and the molecular probes designated TaqMan, FRET, molecular beacons, and variations of these systems. A major advantage of RT-PCR involves the amplification of short DNA target sequences of 60-70-bp, which allows greatly reduced extension times, which when coupled with the advanced technology of RT thermal cyclers with reduced ramp times, greatly reduces cycle times so that amplified targets can be recognized within 30 min of amplification in some cases. Another major advantage is that agarose-gel electrophoresis is not required to visualize amplified target DNA which greatly reduces assay time.

Application of random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PGFE) analysis to Listeria monocytogenes: A review of methodology and results

Robert E. Levin — Thu, 19 Jan 2012 10:24:36 PST

Influence of caffeine on the repair of mitomycin C induced DNA damage

Robert E. Levin et al. — Thu, 19 Jan 2012 10:24:05 PST

Caffeine potentiated MMC induced lethality in Salmonella typhimurium strains proficient in excision and recombinant repair but not in excision or recombinant repair deficient strains. Caffeine was found to inhibit the removal of interstrand DNA crosslinks induced by MMC in strains proficient for excision repair but not in a strain deficient in excision repair. Our results indicate that the potentiation by caffeine of MMC induced lethality in S. typhimurium is primarily due to the inhibition by caffeine of excision repair of interstrand DNA crosslinks.

Application of the polymerase chain reaction for detection of Listeria monocytogenes in foods: a review of methodology

Robert E. Levin — Thu, 19 Jan 2012 10:23:02 PST

The polymerase chain reaction (PCR) as applied to the detection of Listeria monocytogenes in foods encompasses a variety of cell extraction, cell lysis, and DNA purification techniques. These techniques have been applied to foods directly and to enrichment broths with wide variations in sensitivity reported by numerous investigators. The selection of the specific gene for amplification of a selected sequence is also important in that some genes associated with pathogenicity have been found to be present in certain of the other species of Listeria. This review constitutes a detailed presentation of all such factors along with the tabulation of primer sequences, the genes from which they are derived, and the size of the amplified PCR products, in addition to descriptions of the use of nested primers and multiplex PCR.

Quantitative detection of Escherichia coli O157:H7 in ground beef by immunomagnetic separation and competitive polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:18:48 PST

A protocol based on immunomagnetic separation (IMS) and competitive polymerase chain reaction (C-PCR) was developed for quantitative detection of Escherichia coli 0157:H7 in ground beef. Internal standard sequences were synthesized by PCR with hybrid primers composed in part of original primers for C-PCR. A single concentration of internal standards was used for C-PCR after calibration with known amounts of target DNA. After a 6 h enrichment in Tryptic Soy Broth (TSB), C-PCR enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with Shiga-like toxin 1 and 2 (SLT 1 and 2) primers respectively.

Sensitive and rapid detection of Escerichia coli O157:H7 in ground beef by nested PCR incorporating immunomagnetic separation

Robert E. Levin et al. — Thu, 19 Jan 2012 10:18:06 PST

A protocol for detection of low numbers of E. coli O157:H7 in ground beef by nested PCR incorporating immunomagnetic separation (IMS) was developed. The protocol enabled detection of 24 colony-forming-units (CFU) in 10 g of seeded ground beef without enrichment cultivation. Differential centrifugation was used for maximally recovering the target CFU. Partial digestion of the resulting cell pellet with proteinase K at 37°C was used for the removal of beef tissue, which was required for the proper function of IMS. Within the range of 24 to 2400 CFU/10 g, a log linear relationship between the numbers of inoculated CFU and the integrated intensity of the nested PCR products was obtained with both shiga-like toxin (SLT) 1 and 2 primer pairs.

Quantitative detection of Escherichia coli O157:H7 in ground beef by immunomagnetic separation and polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:16:57 PST

A quantitative assay for viable Escherichia coli O157:H7 in ground beef based on immunomagnetic separation (IMS) and the polymerase chain reaction (PCR) was developed. Bacterial cells inoculated into ground beef, were captured by immunomagnetic beads (IMB) after a 6 h non-selective enrichment. The percent capture of the target cells was consistent for the inoculation levels of 0.7 to 70 colony-forming-units (CFU)/g. Captured bacteria were lysed with PCR buffer containing 0.2 mg/mL proteinase K at 65°C for 30 min. DNA sequences of Shiga-like toxin 1 and 2 (Stx 1 and 2) were amplified independently. Log-linear relationships were observed between CFU of E. coli O157:H7 inoculated into ground beef and the integrated fluorescent image of the PCR products with Stx 1 and 2 primers after enrichment. The quantitative range was between 0.7 to 70 CFU/g.

Enhanced storage-life of fresh haddock fillets with stabilized sodium chlorite in ice

Robert E. Levin et al. — Thu, 19 Jan 2012 10:16:10 PST

A method for effectively extending the storage-life of fresh haddock with Salmide® a stabilized form of redox-buffered sodium chlorite in ice was developed. Results from aerobic bacterial counts, trimethylamine (TMA) and odor analyses showed that fresh haddock fillets packed in ice containing 200 ppm sodium chlorite can be stored for about 18 days at 4°C. This storage-life was nearly twice as long as that for fillets packed in ice alone.

Development of a microslide aglutination assay with the aid if an inexpensive projection microscope

Robert E. Levin et al. — Thu, 19 Jan 2012 10:15:13 PST

A microslide agglutination assay was developed involving the mixing of 2.5 microl each of antiserum and a cell suspension of Listeria monocytogenes. Cell agglutination in the final volume of 5.0 microl was visually observed at a direct magnification of 22 x on the projection screen of an inexpensive 20 US dollar projection microscope. The procedure has the advantage of increasing by a factor of 20 the number of agglutination assays that can be performed with a given volume of antiserum with the use of an inexpensive optical projection system.

Quantitative Detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:14:30 PST

A method for quantitative detection of Escherichia coli O157:H7 based on the polymerase chain reaction (PCR) was developed. The method used the NIH Image 1·61 software program to quantitatively analyse the intensity of the fluorescent image of the amplified PCR product. Based on the PCR with SLT1 and SLT2 primers used separately, a log-linear relationship between the numbers of cfu of E. coli O157:H7 inoculated into ground beef and the intensity of the PCR products was achieved with and without enrichment. Without enrichment, 150 cfu of E. coli O157:H7 per gram of ground beef were detected. In contrast, the detection limit decreased to 1·2 cfu g−1 of ground beef using SLT1 and SLT2 primers after 4·5 h of enrichment using modified EC broth with 20 μg ml−1 of novobiocin.

Studies on the growth of Escherichia coli O157:H7 strains at 45oC

Robert E. Levin et al. — Thu, 19 Jan 2012 10:13:17 PST

The objectives of the present report were to examine the ability of 18 strains of Escherichia coli O157:H7 to grow in EC broth at 42.4, 43.5, 44.5, and 45.5°C, and to document the incidence of phenotypic variants present in low numbers that are capable of growth at 45.5°C in EC broth. Among the 18 strains of E. coli O157:H7 studied, only 3 were capable of producing turbid growth with gas formation in EC broth at 45.5°C with 1 × 102 initial CFU/ml. Higher initial densities of CFU resulted in turbid growth and gas formation in EC broth at 45.5° C with all strains. The presence of bile salts #3 in EC broth was found to be inhibitory at 45.5°C. All 18 strains were found to be capable of growth at 45.5°C in nonselective media. The ability of at least one sensitive strain to grow in EC broth at 45.5°C was found to be dependent on the initial number of CFU/ml. Prior growth of cells of a sensitive strain in EC broth at 45.5°C from a cell density of 2.0 × 107 to 8.0 × 107 CFU/ml followed by removal of cells and reinoculation at a cell density of 2.0 × 106 CFU/ml resulted in growth at 45.5°C that did not occur without such conditioning of the inhibitory medium. These results indicate that the ability of most strains of E. coli O157:H7 to grow in EC broth at 45.5°C is dependent on the initial density of CFU and that at low densities of CFU the ability to initiate growth is dependent on either low numbers of phenotypic variants tolerant to the presence of bile salts #3 in EC broth at 45.5°C or to conditioning of the medium with prior elevated numbers of cells.

Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction

Robert E. Levin et al. — Thu, 19 Jan 2012 10:10:21 PST

A new lysis solution designated TZ, consisting of 2.0% Triton X-100 plus 2.5 mg sodium azide/ml in 0.1 M Tris-HCl buffer at pH 8.0, yielded higher levels of genomic DNA from Escherichia coli O157:H7 cells compared with a number of other commonly used cell lysis methods. Ethidium bromide stained DNA bands resulting from PCR amplification of target DNA from 100 CFU of E. coli O157:H7 were readily detected following electrophoresis of agarose gels. In contrast, conventional cell lysis methods failed to detect target DNA from 100 CFU after PCR amplification. The new solution was highly effective for lysing cell suspensions of Salmonella enteritidis, Pseudomonas putida, Lysteria monocytogenes and Psychrobacter immobilis.