Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

Internal sample process control improves cultivation-independent quantification of thermotolerant Campylobacter.

Quantification of Campylobacter is challenging and one major reason is the fact that bacteria lose cultivability due to cold or oxygen stress during storage at retail. Alternative live/dead discriminatory qPCR currently lacks standardization and might overestimate live cells in the presence of dead cells. In this study an internal sample process control (ISPC) was developed. The ISPC consists of a specified number of peroxide-killed C. sputorum cells to be added to each sample in order to monitor (i) the level of reduction of the signal from dead cells and (ii) DNA losses during sample processing. A species-specific fragment of the 16S rRNA gene of C. sputorum was selected as real-time PCR target, based on its similar size and gene copy number compared to the C. jejuni/coli/lari target and confirmed in an exclusivity study. Extension of the amplification oligonucleotides for the target of thermotolerant Campylobacter improved real-time PCR efficiency, rendering the method suitable for quantification according to international standards. Concordant PCR signal variation of both C. jejuni and C. sputorum targets in co-inoculated chicken rinses verified the suitability of the ISPC. This provides a crucial step towards implementation of cultivation-independent quantification for improved food safety of fastidious bacteria.

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.

"Limits of control"--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

A dual approach employing MALDI-TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex.

A real-time PCR procedure targeting the gene of the molecular cochaperon DnaJ (dnaJ) was developed for specific detection of strains belonging to the Enterobacter cloacae group. The inclusivity and exclusivity of the real-time PCR assay were assessed with seven reference strains of E. cloacae, 12 other Enterobacter species and 41 non-Enterobacter strains. Inclusivity as well as exclusivity of the duplex real-time PCR was 100%. In contrast, resolution of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was inadequate for delineation of Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii from E. cloacae. Eleven of 56 (20%) clinical isolates of the E. cloacae group could not be clearly identified as a certain species using MALDI-TOF MS. In summary, the combination of MALDI-TOF MS with the E. cloacae-specific duplex real-time PCR is an appropriate method for identification of the six species of the E. cloacae complex.

Atrazine and terbuthylazine mineralization by an Arthrobacter sp. isolated from a sugarcane-cultivated soil in Kenya.

A tropical soil from a Kenyan sugarcane-cultivated field showed a very high capability to mineralize (14)C-ring-labeled atrazine. In laboratory experiments this soil mineralized about 90% of the applied atrazine within 98 d. The atrazine-degrading microbial community was enriched in liquid cultures containing atrazine as the sole N source and 100 mgL(-1) glucose as additional C source. From the enrichment culture a bacterial strain was isolated and identified by comparative sequence analysis of the 16S-rDNA as member of the genus Arthrobacter. The enriched mixed culture as well as the isolated strain, designated as Arthrobacter sp. strain GZK-1, could grow on atrazine and terbuthylazine as sole N-sources; Arthrobacter sp. GZK-1 mineralized (14)C-ring-labeled atrazine up to 88% to (14)CO(2) and (14)C-ring-labeled terbuthylazine up to 65% to (14)CO(2) in a liquid culture within 14 d. The enriched microbial consortium as well as the isolated strain could be a potential solution for the remediation of s-triazine polluted agricultural soils.