Microbial flora of in-use soap products

A comparison has been made of the in-use bacterial load of two bar soaps with and without antibacterials and two liquid soaps in five different locations over a 1-week period. Of the 25 samples taken from each soap, 92 to 96% of samples from bar soaps were culture positive as compared to 8% of those from liquid soaps. Bacterial populations ranged from 0 to 3.8 log CFU per sample for bar soaps and from 0 to 2.0 log CFU per sample for liquid soaps. The mean bacterial populations per sample were 1.96 and 2.47 log CFU for the two bar soaps, and 0.08 and 0.12 log CFU for the two liquid soaps. The difference in bacterial population between bar soaps and liquid soaps was statistically significant (P = 0.005). Staphylococcus aureus was isolated on three occasions from bar soaps but not from liquid soaps. S. aureus was isolated twice from the exterior of the plastic dispensers of liquid soap but not from the soap itself. Gram-negative bacteria were cultured only from soaps containing antibacterials. Bacterial populations on bar soaps were not high compared with bacterial populations on hands, and the flora was continually changing without evidence of a carrier state.     

Bacterial hand contamination and transfer after use of contaminated bulk-soap-refillable dispensers

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log(10) CFU/ml of Serratia marcescens, an average of 5.28 log(10) CFU remained on each hand after washing, and 2.23 log(10) CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log(10) CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log(10) CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log(10) CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log(10) CFU; P < 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log(10) CFU per hand) than were recovered from staff (2.22 log(10) CFU per hand) after washing with contaminated bulk soap (P < 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.     

Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions

Antimicrobial hand soaps provide a greater bacterial reduction than nonantimicrobial soaps. However, the link between greater bacterial reduction and a reduction of disease has not been definitively demonstrated. Confounding factors, such as compliance, soap volume, and wash time, may all influence the outcomes of studies. The aim of this work was to examine the effects of wash time and soap volume on the relative activities and the subsequent transfer of bacteria to inanimate objects for antimicrobial and nonantimicrobial soaps. Increasing the wash time from 15 to 30 seconds increased reduction of Shigella flexneri from 2.90 to 3.33 log(10) counts (P = 0.086) for the antimicrobial soap, while nonantimicrobial soap achieved reductions of 1.72 and 1.67 log(10) counts (P > 0.6). Increasing soap volume increased bacterial reductions for both the antimicrobial and the nonantimicrobial soaps. When the soap volume was normalized based on weight (approximately 3 g), nonantimicrobial soap reduced Serratia marcescens by 1.08 log(10) counts, compared to the 3.83-log(10) reduction caused by the antimicrobial soap (P < 0.001). The transfer of Escherichia coli to plastic balls following a 15-second hand wash with antimicrobial soap resulted in a bacterial recovery of 2.49 log(10) counts, compared to the 4.22-log(10) (P < 0.001) bacterial recovery on balls handled by hands washed with nonantimicrobial soap. This indicates that nonantimicrobial soap was less active and that the effectiveness of antimicrobial soaps can be improved with longer wash time and greater soap volume. The transfer of bacteria to objects was significantly reduced due to greater reduction in bacteria following the use of antimicrobial soap.     

The effect of a commercial UV disinfection system on the bacterial load of shell eggs

AIMS: To study the effect of UV irradiation on the bacterial load of shell eggs and of a roller conveyor belt. METHODS AND RESULTS: The natural bacterial load on the eggshell of clean eggs was significantly reduced by a standard UV treatment of 4.7 s; from 4.47 to 3.57 log CFU per eggshell. For very dirty eggs no significant reduction was observed. Eggs inoculated with Escherichia coli and Staphylococcus aureus (4.74 and 4.64 log CFU per eggshell respectively) passed the conveyor belt and were exposed to UV for 4.7 and 18.8 s. The reduction of both inoculated bacteria on the eggshell was comparable and significant for both exposure times (3 and 4 log CFU per eggshell). Escherichia coli was reduced but still detectable on the conveyor rollers. The internal bacterial contamination of eggs filled up with diluent containing E. coli or S. aureus was not influenced by UV irradiation. Conclusions: There is a significant lethal effect of UV irradiation on the bacterial contamination of clean eggshells and recent shell contamination, contamination of rollers can be controlled and the internal contamination of eggs is not reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: The penetration of UV into organic material appears to be poor and UV disinfection can be used as an alternative for egg washing.     

Occurrence of foodborne pathogenic bacteria in retail prepackaged portions of marine fish in Spain

AIMS: To survey the presence of indigenous and nonindigenous foodborne bacterial pathogens in displayed prepacked portions of fresh marine fish. METHODS AND RESULTS: A survey of 50 different samples of fresh marine fish (conger, swordfish, sole, grouper and whiting) was conducted over a period of 5 months. Trays of fillets and steaks were obtained at retail level and tested for foodborne bacterial pathogens. Vibrio cholerae and Salmonella were not detected. Two samples (4%) yielded Vibrio strains carrying a DNA fragment specific for Vibrio parahaemolyticus, but resulted negative to PCR amplification of the virulence-related tdh gene. Levels of motile Aeromonas ranging from 2.29 to 7.20 log CFU g(-1) were found in 31 (62%) samples. All fish portions were positive for the Aeromonas hlyA gene and 38 for both aerA and hlyA genes, which may contribute to diarrhoea-related virulence. The incidence of Listeria monocytogenes was 10%. Levels of Staphylococcus aureus lower than 2 log CFU g(-1) were found in 15 (30%) samples. Numbers of presumptive Clostridium perfringens ranging from 1.82 +/- 0.22 to 4.26 +/- 1.25 log CFU g(-1) were detected in 42 (84%) samples. Edwardsiella tarda was detected in two samples of grouper fillets. CONCLUSIONS: Displayed portions of raw fish carried bacteria that can cause foodborne disease. The risk posed by fresh fish when properly cooked is low, but high when destined to be consumed raw, undercooked or very lightly processed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that raw fish sold in Spain could be a source of foodborne bacterial pathogens. Improvements in handling and processing are needed to minimize the prevalence of pathogenic bacteria.     

Evaluation and remediation of bulk soap dispensers for biofilm

Recent studies evaluating bulk soap in public restroom soap dispensers have demonstrated up to 25% of open refillable bulk-soap dispensers were contaminated with ~ 6 log(10)(CFU ml(-1)) heterotrophic bacteria. In this study, plastic counter-mounted, plastic wall-mounted and stainless steel wall-mounted dispensers were analyzed for suspended and biofilm bacteria using total cell and viable plate counts. Independent of dispenser type or construction material, the bulk soap was contaminated with 4-7 log(10)(CFU ml(-1)) bacteria, while 4-6 log(10)(CFU cm(-2)) biofilm bacteria were isolated from the inside surfaces of the dispensers (n = 6). Dispenser remediation studies, including a 10 min soak with 5000 mg l(-1) sodium hypochlorite, were then conducted to determine the efficacy of cleaning and disinfectant procedures against established biofilms. The testing showed that contamination of the bulk soap returned to pre-test levels within 7-14 days. These results demonstrate biofilm is present in contaminated bulk-soap dispensers and remediation studies to clean and sanitize the dispensers are temporary.     

Rapid and direct detection of Invivo kinetics of pathogenic bacterial infection from mouse blood and urine

This study demonstrates the first use of matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to trace the Invivo infection kinetics of the well known deadly pathogen Staphylococcus aureus in Swiss albino mice. The growth curve of the bacteria from the point of injection (200μL of bacterial suspension (10(8)cfu/mL)) into the mouse blood till mortality (death) was periodically analyzed using the plate counting method and MALDI-MS. Bacterial counts of 10(3)cfu/mL were observed in the log phase of the growth curve in the blood and 10(2)cfu/mL were observed in the urine samples. Death occurred in the log phase of the growth curve, where the bacterial counts showed steady increase. In other cases, the bacteria counts started decreasing after 48h and by 96h the bacteria got totally eliminated from the mouse and these mice survived. Direct MALDI-MS was not feasible for tracking the bacteria in the infected blood. However, ionic liquid 1-Butyl-3-methylimidazolium tetrafluoroborate was successful in enabling bacterial detection amidst the strong blood peaks. But, in the case of the urine analysis, it was observed that direct MALDI-MS was adequate to enable detection. The results obtained prove the efficacy of MALDI-MS for analyzing pathogenic bacteria in clinical samples. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Effect of sampling scale on the assessment of epiphytic bacterial populations

Bacterial populations on above-ground plant surfaces were estimated at three different biological scales, including leaflet disks, entire leaflets, and whole plants. The influence of sample scale on the estimation of mean bacterial population size per unit and per gram and on the variability among sampling units was quantified at each scale. Populations were highly variable among sampling units at every scale examined, suggesting that there is no optimal scale at which sample variance is reduced. The distribution of population sizes among sample units was sometimes, but not consistently, described by the lognormal. Regardless of the sampling scale, expression of population sizes on a per gram basis may not reduce variance, because population size was not generally a function of sample unit weight within any single sampling scale. In addition, the data show that scaling populations on a per gram basis does not provide a useful means of comparing population estimates from samples taken at different scales. The implications of these results for designing sampling strategies to address specific issues in microbial ecology are discussed. Correspondence to: L.L. Kinkel     

Bacterial Responses to Different Dietary Cereal Types and Xylanase Supplementation in the Intestine of Broiler Chicken

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4- g - glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.     

Glioperazine B, as a new antimicrobial agent against Staphylococcus aureus, and glioperazine C: two new dioxopiperazines from Bionectra byssicola

In the course of our screening for new antibacterials from microbial metabolites, two new dioxopiperazine metabolites, glioperazines B (1) and C (2), together with the known compound, glioperazine (3), were isolated from the mycelia of a liquid fermentation culture of the fungus, Bionectra byssicola F120. The structures of compounds 1 and 2 were assigned on the basis of MS and NMR data. Compound 1 is an unusual dioxopiperazine metabolite containing an OMe group at the alpha-carbon of the amino acid residue. Compound 1 showed weak antibacterial activity against various strains of S. aureus, including methicillin-resistant S. aureus (MRSA) and quinolone-resistant S. aureus (QRSA), while 2 and 3 did not show such activity.     

Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage

AIMS: To determine the effectiveness of an alkaline cleaner used in food-processing plants and a lytic bacteriophage specific for Escherichia coli O157:H7 in killing wild type and rpoS-deficient cells of the pathogen in a biofilm. METHODS AND RESULTS: Wild type and rpoS-deficient cells were attached to stainless steel coupons (c. 7-8 log CFU per coupon) on which biofilms were developed during incubation at 22 degrees C for 96 h in M9 minimal salts media (MSM) with one transfer to fresh medium. Coupons were treated with 100 and 25% working concentrations of a commercial alkaline cleaner (pH 11.9, with 100 microg ml(-1) free chlorine) used in the food industry, chlorine solutions (50 and 100 microg ml(-1) free chlorine), or sterile deionized water (control) at 4 degrees C for 1 and 3 min. Treatment with 100% alkaline cleaners reduced populations by 5-6 log CFU per coupon, a significant (P < or = 0.05) reduction compared with treatment with water. Initial populations (2.6 log CFU per coupon) of attached cells of both strains were reduced by 1.2 log CFU per coupon when treated with bacteriophage KH1 (7.7 log PFU ml(-1)) for up to 4 days at 4 degrees C. Biofilms containing low populations (2.7-2.8 log CFU per coupon) of wild type and rpoS-deficient cells that had developed for 24 h at 22 degrees C were not decreased by more than 1 log CFU per coupon when treated with KH1 (7.5 log PFU ml(-1)) at 4 degrees C. CONCLUSIONS: Higher numbers of cells of E. coli O157:H7 in biofilms are killed by treatment with an alkaline cleaner than with hypochlorite alone, possibly through a synergistic mechanism of alkaline pH and hypochlorite. Populations of cells attached on coupons were reduced by treating with bacteriophage but cells enmeshed in biofilms were protected. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline pH, in combination with hypochlorite, in a commercial cleaner is responsible for killing E. coli O157:H7 in biofilms. Treatment with bacteriophage KH1 reduces populations of cells attached to coupon surfaces but not cells in biofilms.     

Influence of apple cultivars on inactivation of different strains of Escherichia coli O157:H7 in apple cider by UV irradiation

This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6) to 10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm(2). Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (alpha < or = 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (alpha < or = 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of (o)Brix, pH, and malic acid content failed to show any statistically significant relationship (R(2) > or = 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested.     

High phenotypic diversity in infecting but not in colonizing Staphylococcus aureus populations

In hostile environments diversity within a bacterial population may be beneficial for the fitness of the microbial community as a whole. Here we analysed the population diversity of Staphylococcus aureus in infecting and colonizing situations. In the study, performed independently in two German centres, the heterogeneity of the S. aureus population was determined by quantifying the occurrence of phenotypic variants (differences in haemolysis, pigmentation, colony morphology) in primary cultures from nose, oropharyngeal and sputum specimens from cystic fibrosis (CF) patients and in nose swabs from healthy S. aureus carriers. The proportion of heterogeneous samples, the number of clearly distinguishable isolates per sample and the qualitative differences between phenotypes was significantly higher in CF sputum specimens than in the other samples. The heterogeneity of the S. aureus population could be correlated with high bacterial densities in the sputum samples. In patients co-infected with Pseudomonas aeruginosa lower S. aureus bacterial loads and less heterogeneity in the S. aureus population were observed. Typing of all S. aureus isolates from heterogeneous samples by pulsed-field gel electrophoresis or spa typing revealed that the bacteria were polyclonal in 30%, monoclonal with minor genetic alterations in 25% or not distinguishable in 69% of the specimens. Some specimens harboured monoclonal and polyclonal variants simultaneously. Importantly, differences in antibiotic susceptibility were detected in phenotypic S. aureus variants within a single specimen. Diversification of a S. aureus population is highly favoured during chronic CF lung infection, supporting the general hypothesis that maintenance of intrahost diversity can be of adaptive value, increasing the fitness of the bacterial community.     

LOW NUTRIENT R2A CULTURE MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS

Poultry feed matrices can be particularly stressful to bacterial populations and limit recovery and accurate enumeration of viable organisms. The objectives in this study were to enumerate bacterial populations with either tryptic soy plate medium or an R2A minimal nutrient medium from commercial poultry and turkey dry feeds and compare enumerations from the two media with feed composition. Total population estimates from tryptic soy plates varied between 3.7 and log₁₀ 5.4 CFU per g feed and depended upon poultry feed source (P < 0.05). R2A plate counts varied between 2.7 and log₁₀ 5.4 CFU per g feed, but were not significantly different among poultry feed sources. Feed R2A plate count populations were significantly correlated (P < 0.01) with tryptic soy plate count populations enumerated from the feed sources, but not protein and fat content of the feed source. It is apparent that bacterial counts recovered by minimal R2A medium are similar to enumerations using tryptic soy plate medium and that R2A medium could be substituted for tryptic soy plate medium for bacterial enumeration in poultry feeds.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Comparing antibiotic resistance in commensal and pathogenic bacteria isolated from wild-caught South Carolina shrimps vs. farm-raised imported shrimps

The objective of this study was to assess and differentiate wild-caught South Carolina (SC) shrimps from imported shrimps on the basis of microbiological analysis. Seven wild-caught SC shrimp and 13 farm-raised imported shrimp samples were analyzed. Total plate counts from wild-caught shrimp samples ranged from 4.3 to 7.0 log10 CFU/g, whereas counts from imported shrimp samples ranged from 3.2 to 5.7 log10 CFU/g. There was no difference (P > 0.05) between total bacterial counts of wild-caught SC shrimp and farm-raised imported shrimp. However, the percentages of bacteria with reduced susceptibility towards ceftriaxone and tetracycline were higher (P < 0.05) for farm-raised shrimp than for wild-caught samples. Salmonella spp. detected only in one farm-raised sample was resistant to ampicillin, ceftriaxone, gentamicin, streptomycin, and trimethoprim. Vibrio vulnificus was detected in both wild-caught and farm-raised shrimp samples; however, only the isolate from farm-raised shrimp was resistant to nalidixic acid and trimethoprim. Escherichia coli detected in one wild-caught sample was resistant to ampicillin. Both Listeria spp. and Salmonella spp. were absent with wild-caught SC samples. Therefore, the presence of more ceftriaxone- and tetracycline-resistant bacteria and the observed antimicrobial resistance phenotypes of isolates from the imported shrimp may reflect the possible use of antibiotics in raising shrimp in those countries.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Corneal virulence of Staphylococcus aureus in an experimental model of keratitis

The aim of this study was to determine the pathogenic role of alpha-, beta-, and gamma-toxins in a rabbit model of Staphylococcus aureus keratitis. S. aureus strains 8325-4, Newman, and their isogenic mutants were intrastromally injected into rabbit corneas. Eyes were scored for pathology by slit lamp examination (SLE), histologic examination, and bacterial colony-forming units (CFU) per cornea were determined. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain 8325-4 or Newman. All strains grew equivalently to approximately 7 log CFU/cornea at 25 h postinfection. SLE scores at 15, 20, and 25 h postinfection revealed that alpha-toxin - producing strains caused greater corneal pathology than strains deficient in alpha-toxin. A beta-toxin - deficient mutant produced significantly less ocular edema than its parent or rescued strains. The gamma-toxin-deficient mutant, relative to its parent strain or genetically rescued strain, had reduced virulence. These results demonstrate that the virulence of S. aureus involves mainly alpha-toxin and to a lesser extent gamma-toxin, with beta-toxin mediating minimal corneal pathology.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.