Ingeborg Hein

Comparison of Different Approaches To Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

ABSTRACT Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures ofS. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 × 102 to 6.4 × 102 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Survey on the Listeria contamination of ready-to-eat food products and household environments in Vienna, Austria.

Qualitative and quantitative contamination of ready‐to‐eat food‐stuffs with the pathogen Listeria monocytogenes was studied in 1586 samples collected from 103 supermarkets (n = 946) and 61 households (n = 640) in Vienna, Austria. Seventeen groups of ready‐to‐eat foods were classified into three risk categories for contamination (CP1–CP3). Three to four samples were randomly collected at the retail level from each CP. Regarding the households, the sampling procedure was started with food items of CP1, and if not available, was continued with sampling of food items of CP2 and finally of CP3. Additionally, 184 environmental samples (swabs from the kitchen area, dust samples from the vacuum cleaner) and faecal samples (household members and pet animals) were included. One‐hundred and twenty‐four (13.1%) and 45 (4.8%) samples out of 946 food samples collected from food retailers tested positive for Listeria spp. and L. monocytogenes, respectively, with five smoked fish samples exceeding the tolerated limit of 100 CFU/g food. Food‐stuffs associated with the highest risk of contamination were twice as frequently contaminated with L. monocytogenes as food‐stuffs associated with a medium risk of contamination. Products showing the highest contamination rate were fish and seafood (19.4%), followed by raw meat sausages (6.3%), soft cheese (5.5%) and cooked meat products/patés (4.5%). The overall contamination rate of foods collected at the household level was more than two times lower. Only 5.6% and 1.7% of 640 food‐stuffs analysed tested positive for Listeria spp. and L. monocytogenes, respectively. However, CP1 foods were rarely collected. Pulsed‐field gel electrophoresis (PFGE) typing of the collected L. monocytogenes isolates revealed a high degree of diversity between the isolates, with some exceptions. PFGE typing of isolates harvested from green‐veined cheese revealed a match among strains, although the manufacturer seemed to be distinguishable. Typing of household strains revealed an epidemiological link within one family. In this case, food‐stuffs and the kitchen environment were contaminated by an indistinguishable isolate. In addition, the same isolate was collected from a pooled faecal sample of the household members suggesting that consumption of even low contaminated food items (<100 CFU/g) results in Listeria shedding after the passage through the gut.

Evaluation of Paramagnetic Beads Coated with Recombinant Listeria Phage Endolysin–Derived Cell-Wall-Binding Domain Proteins for Separation of Listeria monocytogenes from Raw Milk in Combination with Culture-Based and Real-Time Polymerase Chain Reaction–Based Quantification

The aim of this study was to evaluate a fast and simple bead-based method using paramagnetic beads covered with recombinant Listeria phage endolysin-derived cell-wall-binding domain proteins specific for Listeria spp. for separation of the foodborne pathogen Listeria monocytogenes from artificially contaminated raw milk. The method was combined with subsequent detection and quantification by the traditional plate-count technique and real-time polymerase chain reaction (PCR). To account for differences in cell properties, recovery rates and the detection limit were determined using five different L. monocytogenes strains for preparation of a 10-fold dilution series in raw milk, spanning an 8-log scale. Two independent test series were performed for each strain, yielding mean recovery rates of 46.6% to 122.8% for detection with the plate-count method, and 64.7% to 95.1% for detection by real-time PCR. A high correlation was found between the number of L. monocytogenes added to the samples and the number of colony forming units recovered by plate count (0.980), as well as the number of bacterial cell equivalents obtained by real-time quantitative PCR (0.987). The detection limit of the combined cell-wall-binding domain proteins/real-time PCR approach ranged from 10(2) to 10(3) colony forming units per milliliter, which is close to the theoretical detection limit of the method.

Stress- and Growth Rate-Related Differences between Plate Count and Real-Time PCR Data during Growth of Listeria monocytogenes

ABSTRACT To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10°C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r2 = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r2 = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.

Stress Survival Islet 1 (SSI-1) Survey in Listeria monocytogenes Reveals an Insert Common to Listeria innocua in Sequence Type 121 L. monocytogenes Strains

ABSTRACT Listeria monocytogenes strains (n = 117) were screened for the presence of stress survival islet 1 (SSI-1). SSI-1+ strains (32.5%) belonged mainly to serotypes 1/2c, 3b, and 3c. All sequence type 121 (ST-121) strains included (n = 7) possessed homologues to Listeria innocua genes lin0464 and lin0465 instead of SSI-1.

Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR.

Comparison of a broad range of characteristics of real-time PCR amplification curves yielded only slight alterations for low numbers of mismatches in the primer binding regions, resulting in a quantification error up to 63.12%. The effects were more pronounced for mismatches in the probe binding region and resulted in a quantification error up to 33%.

Phenotypic and molecular characterization of Staphylococcus aureus strains of veterinary, dairy and human origin

Austrian veterinary (n=91), dairy (n=86), and human strains (n=48) of Staphylococcus aureus were tested for various phenotypic properties including clumping factor, egg-yolk reaction, production of thermonuclease and susceptibility to 14 antibiotics. In addition the expression of enterotoxins (A-E), and the presence of enterotoxin genes sea to sej and tst was determined. Significant differences in antimicrobial susceptibility were found with 84.6% of veterinary, 57.0% of dairy, and 20.8% of human strains susceptible to all antibiotics tested (P<0.0005). More human strains produced enterotoxins (41.7%) than veterinary (9.9%) and dairy strains (12.6%) while 40.7% and 38.5% of veterinary, 47.7% and 52.3% of dairy, and 77.1% and 87.5% of human strains were se- and tst-positive, respectively. AFLP analysis revealed nine clusters with over- or under-representation of strains with specific characteristics. Strains clustered according to origin (veterinary, dairy, and human) and/or presence of toxin genes and antimicrobial resistance.

Characterization of Listeria monocytogenes isolates from 50 small-scale Austrian cheese factories.

One hundred eighty-one small-scale cheese factories (annual production < 100,000 kg) were tested for the presence of Listeria monocytogenes in cheese and smear samples from 1997 to 2000. In total, 2615 samples were drawn. Fifty (27.6%) of 181 enterprises yielded L. monocytogenes. From 14 of the cheese-making facilities, we obtained more than four L. monocytogenes isolates. A total of 182 mostly cheese- and smear-borne L. monocytogenes strains were characterized by serotyping and pulsed-field gel electrophoresis. In 12 of 14 cheese factories, over half of the L. monocytogenes isolates were genetically indistinguishable by pulsetype. On average, genetically indistinguishable isolates were recovered for 11.9 months. Regarding serotypes, 27.3% of the isolates were of serovar 4b. Inadequate personal hygiene could explain the high prevalence of serovar 4b isolates in small-scale cheesemaking facilities. Forty-two percent of the serovar 4b isolates recovered from epidemiologically unlinked facilities (in comparison to 40 and 29% of the 1/2a and 1/2b isolates, respectively) were genetically indistinguishable from at least one other isolate. Indistinguishable serovar 1/2a and 1/2b isolates belonged to five and six different pulsetypes, respectively, whereas serovar 4b isolates belonged to only two pulsetypes. This finding suggested a wide distribution of genetically homologous serovar 4b isolates among the facilities tested in our study.

Novel Real-Time PCR Assay for Simultaneous Detection and Differentiation of Clostridium chauvoei and Clostridium septicum in Clostridial Myonecrosis

ABSTRACT A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 × 103 C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method.