Roles of viable but non-culturable state in the survival of Campylobacter jejuni

Abstract

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Detection of Viable but Non-Culturable (VBNC)-Campylobacter in the Environment of Broiler Farms: Innovative Insights Delivered by Propidium Monoazide (PMA)-v-qPCR Analysis

“…Interestingly, storing milk-induced VBNC C. jejuni in water resulted in maintenance of viable C. jejuni counts during the experimental period of 25 days in habitats A and B and even 109 days in habitat C. This may imply that stress conditions in water have less impact on VBNC stability than those encountered in soil [33,48]. These findings are consistent with prior research that has examined VBNC Campylobacter in both water-and food-related matrices under comparable conditions [20][21][22]33].…”

“…Thus, employing v-qPCR in conjunction with ISPC could offer additional insights into the presence of VBNC-Campylobacter in aquatic farm environments, as previously indicated [53]. The urgency for this is further supported by recent findings where the virulence of VBNC C. jejuni in primary chicken embryo cells (accordingly outgoing pathogenicity) was demonstrated [21]. As described earlier, at the farm level, VBNC C. jejuni may persist in manure residues or contaminations in the environment.…”

“…Moreover, it has been suggested that the availability of organic matter (manure remnants) in water bodies provides ample nutrients that allow Campylobacter persistence [35]. Yet, it can be contended that nutritional and oxidative stress also promote rapid VBNC induction, as observed in a recent study [21]. In this study, most viable C. jejuni were primarily detected by v-qPCR after treatment with PMA in sock swab samples (45.8%), collected by walking a predetermined route at Campylobacter-positive farms.…”

“…The persistence of Campylobacter in the environment of broiler farms and subsequent colonization of broilers may also be related to the VBNC state. Previous studies have demonstrated that VBNC-Campylobacter (C. jejuni) can resuscitate in vivo or under laboratory conditions, as well as express pathogenicity [11,20,21]. Furthermore, recent research has underscored the importance of VBNC Campylobacter in food processing conditions.…”

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni

“…When C. jejuni is subjected to environmental stresses such as low temperature, high osmotic stress, nutrient starvation, or unsuitable aerobic conditions, it enters a viable but non-culturable (VBNC) state (Yagi et al 2022 ). In the VBNC state, C. jejuni is transformed from the normal spiral form into a coccoid form under unfavourable environmental conditions (Santos et al 2023 ). Despite the lack of clear evidence to prove that the VBNC state of C. jejuni can directly cause human diseases, the risks to food safety and public health cannot be ignored (Chaisowwong et al 2012 ).…”