Chicken Skin Decontamination of Thermotolerant Campylobacter spp. and Hygiene Indicator Escherichia coli Assessed by Viability Real-Time PCR.

Thermotolerant Campylobacter spp. are fecal contaminants of chicken meat with serious implications for human health. E. coli is considered as hygiene indicator since, in contrast to Campylobacter. spp., the bacterium is generally present in the avian gut. Stress exposure may transiently cease bacterial division. Therefore, colony forming units (CFU) may underestimate the infection risk of pathogens. We developed a viability real-time PCR (v-qPCR) for the quantification of viable E. coli targeting the uidA gene, encoding ß-glucuronidase, which is usually detected for phenotypic species identification. The short- and long-term effects of decontaminating chicken skin on the survival of both C. jejuni and an ESBL-producing E. coli were evaluated by CFU and v-qPCR. The results showed that freezing and storage in cool conditions are potentially underestimated by CFU but not by v-qPCR. The effect of treatment with peroxyacetic acid on survival was consistently detected by CFU and v-qPCR. v-qPCR analysis detected bacterial survival upon the application of lactic acid, which awaits further analysis. Interestingly, both bacteria showed similar kinetics of inactivation upon the application of reduction strategies, suggesting that E. coli might be a complementary hygiene indicator. We conclude that v-qPCR can improve food safety under the consideration of some limitations.

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.

Underestimated Survival of Campylobacter in Raw Milk Highlighted by Viability Real-Time PCR and Growth Recovery.

Raw milk is a frequent vehicle for transmission of thermophilic Campylobacter, leading to reported outbreaks. Milk is a challenging food matrix for pathogen detection, due to its high protein and lipid content. Limited detection of Campylobacter colony-forming unit (CFU) in raw milk might underestimate the pathogen's infectious potential. We optimized a viability real-time PCR (qPCR) for application with raw milk. The procedure was robust against variations of milk lots and different Campylobacter strains. Various DNA-intercalating dyes were evaluated for their ability to reduce the PCR signal of dead cells. Only propidium monoazide (PMA) and PMAxx qualified for diagnostic use. Different sedimentation properties of viable and dead Campylobacter jejuni and Campylobacter coli strains in 10-fold diluted milk enhanced viable/dead differentiation. The new method enabled to review survival of Campylobacter spp. in raw milk based on viable cells harboring an intact cell membrane. The data were compared to culturability according to ISO10272-2:2017. A difference of up to 4.5 log10 between viable Campylobacter counts and CFU values became apparent. Relevance of viability qPCR values was corroborated by full recovery of CFU under extremely reduced oxygen concentration in the presence of hydrogen. Recovery of CFU was limited, however, upon prolonged exposure in raw milk. The data confirm that Campylobacter survival in raw milk can be largely underestimated when relying on CFU data only. We conclude that raw milk led to oxidative stress-induced growth arrest in thermophilic Campylobacter, which was reversible by reduction of the oxygen partial pressure in a time-limited way.

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.

Investigation of the Anti-Leishmania (Leishmania) infantum Activity of Some Natural Sesquiterpene Lactones.

Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus Leishmania. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.