BackgroundThermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR.ResultsSpecies specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low.ConclusionsProposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.

Quorum sensing is a widespread form of cell-to-cell communication, which is based on the production of signaling molecules known as autoinducers (AIs). The first group contains highly species-specific N-acyl homoserine lactones (N-AHLs), generally known as AI-1, which are produced by AHL synthase. The second group, possessing the characteristic structure of a furanone ring, are known as AI-2. The enzyme responsible for their production is S-ribosylhomocysteine lyase (LuxS). In Campylobacter jejuni, AI-2 and LuxS play a role in many important processes, including biofilm formation, stress response, motility, expression of virulence factors, and colonization. However, neither the receptor protein nor the exact structure of the AI-2 molecule have been identified to date. Similarly, little is known about the possible existence of AHL-synthase producing AI-1 and its impact on gene expression. Recently, an analogue of homoserine lactone, called cjA, was isolated from a cell-free supernatant of C. jejuni strain 81–176 and from the food isolate c11. The molecule cjA particularly impacted the expression of virulence factors and biofilm formation. This review summarizes the role of AI-2 and cjA in the context of biofilm formation, motility, stress responses, and expression of virulence factors.

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Currently, it is clear that the luxS gene has an impact on the process of bio lm formation in Campylobacter jejuni. However, even within the species naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form bio lms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in bio lm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Bio lm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed signi cantly less bio lm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in bio lm mass than the substitution (•luxS) and insertional inactivated ( luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the bio lm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex bio lm, and to develop microcolonies at the level of the wild-type.

Vondráková L., Purkrtová S., Pazlarová J., Demnerová K. (2015): Species differentiation of thermotolerant Campylobacters based on distinctive banding patterns obtained by multiplex PCR. Czech J. Food Sci., 33: 27-31.The differentiation of thermotolerant Campylobacter spp. on the species level (C. jejuni, C. coli, C. lari, and C. upsaliensis) was provided. Identification is based on different banding patterns obtained for individual species during simple multiplex PCR where regions within the 23S rRNA gene are amplified using newly designed specific forward primers.

Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81–176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated (⸬luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.