Comparison between conventional and qPCR methods for enumerating Campylobacter jejuni in a poultry processing plant

Campylobacter jejuni is worldwide recognized as a human foodborne pathogen. It is widely present in poultry meat and slaughterhouses, but little is known about its fate during the processing of poultry meat preparations. In stress conditions, this pathogen can enter into a viable but non-culturable state, where quantitative PCR (qPCR) becomes more convenient for its detection. In this study, two different pairs of primers, targeting the rpoB and the hipO genes, were compared for its detection and quantification by PCR. Two calibration curves were prepared: one for the meat samples and the other for the environmental samples. rpoB primers showed higher sensitivity with a quantification limit of 1 log cfu/g or ml. Microbial Assessment Scheme (MAS) was used to select the Critical Sampling Locations (CSLs) along the poultry processing line. Forty-six out of 48 samples were positive by qPCR after enrichment (t = 48 ) while only 6 samples were positive by ISO 10272-1:2006. Forty-three samples showed positive signal without enrichment (t = 0 h), however only 16 samples could be quantified. These results showed the high prevalence of C. jejuni in the poultry industry and the need for new, rapid and sensitive techniques, such as qPCR, for the detection and quantification of C. jejuni in meat and environmental samples.     

Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 10³ cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.     

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼10⁵ to 10⁶ cfu cm⁻²C. jejuni, and were stored under aerobic or vacuum packaged conditions at −1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm⁻² reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.     

A survey on the incidence of Campylobacter spp. and the development of a surface adhesion polymerase chain reaction (SA-PCR) assay for the detection of Campylobacter jejuni in retail meat products

A rapid and sensitive method for the detection of Campylobacter in meat products was developed.Campylobacter were isolated from a range of Irish retail meats (n=80) and poultry (n=100) samples by direct plating on Campylobacter Selective Agar (CCDA, Oxoid). A total of 30·5% of samples tested positive forCampylobacter and Campylobacter jejuni was the most commonly identified species. The pathogen was detected in 39 (65%) poultry, 12 (60%) offal and 4 (20%) minced beef samples examined. Estimates derived from direct plate counts ranged from log₁₀0·7 to log₁₀2·75 cfu g⁻¹. The data from this study was used in the development of a rapid and sensitive method for the detection of Campylobacter in poultry. A rapid method was developed based on an initial sample enrichment for 24 h in Campylobacter Enrichment Broth (CEB), recovery of the pathogen from the enriched sample by surface adhesion onto a polycarbonate membrane, phenol:chloroform extraction of DNA from the adherent bacteria, and PCR analysis using primers specific for the flagellin A gene (present in C. jejuni and C. coli). The developed surface adhesion PCR (SA-PCR) technique had a detection limit of log₁₀4–5 and could be completed within 29 h. Results from SA-PCR analysis of a number of retail samples (n=50) correlated favourably with traditional plate culture results, i.e. 34 samples were found to contain Campylobacter by both methods.     

Resistance of Campylobacter jejuni Isolated from Layer Farms in Northern Jordan Using Microbroth Dilution and Disc Diffusion Techniques

Campylobacter jejuni is an important pathogen of significant public health importance. This pathogen is associated with human infection and has been isolated from mammals and birds. Ninety‐two cloacal C. jejuni isolates were identified from 35 layer farms in Northern Jordan. Antimicrobial susceptibility was determined using minimal inhibitory concentration (MIC) and disc diffusion techniques with variable suggested breakpoints. Using MIC and EUCAST cut‐off values, the study revealed a significantly high resistance level (100%) among the layers’ isolates against ciprofloxacin and tetracycline. A relatively high resistance (41%) toward gentamicin and amoxicillin and low resistance to nalidixic acid (21%), erythromycin (14%), and florfenicol (6.5%) were also found. This high level of resistance may indicate abuses in the handling of antibiotics, which may require stricter control in the local layer industry. A good agreement between the 2 techniques used was demonstrated and the disc diffusion technique could be used as a rapid screening test for antimicrobial susceptibility of C. jejuni to many antibiotics using specific Campylobacter breakpoints.     

Prevalence and survival of Campylobacter in Egyptian dairy products

Campylobacter is a leading foodborne pathogen that poses an interesting paradox of being frequently transmitted by foodstuffs, yet showing sensitivity to food preservation procedures. In the present study, 227 samples of milk and dairy products were randomly collected and examined for the presence of Campylobacter spp. Samples were collected from the Egyptian area Abou-Homos, where the pathogen had been previously shown to be a major causative agent of intestinal disease. Potential Campylobacter isolates were speciated using a multiplex PCR assay targeting the housekeeping gene lpxA. Only raw milk and fresh Domiati cheese samples were found to contain Campylobacter jejuni at low incidence rates. Using a selected C. jejuni isolate recovered in this study, it was shown that the pathogen maintained its viability in Domiati cheese more than in yoghurt, yet it survived in both products more than expected. This suggests that this foodborne strain of C. jejuni may develop adaptive strategies that aid survival under food preservation conditions, which contradicts with what is known about this pathogen as a stress-sensitive organism.     

Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

Recovery of viable but non-culturable Campylobacter jejuni cells in two animal models

As in many bacteria, including such pathogens as Salmonella enteritidis, enterotoxicEscherichia coli, Vibrio vulnificus, Vibrio cholerae , the occurrence of a viable but non-culturable (VBNC) state has been described for Campylobacter jejuni. This bacteria has been recognized as a leading foodborne pathogen. In this study, VBNC cells from three C. jejuni human isolates were suspended in microcosm water at 4°C and entered the VBNC state. The metabolic activity of these VBNC cells was monitored by CTC reduction. Once in VBNC state, the strains were inoculated per os into newborn mice and 1-day-old chicks so as to evaluate and compare these animal models of VBNC cell recovery. The three strains used in this experiment were revived using the murine model, whereas only two strains were revived with the chick model. The results show that the murine model permits better recovery than the 1-day chick model. All three strains revived exhibited an associative index very similar to that measured in the culturable state. This study indicates that the VBNC state should be considered as playing a role in the epidemiology of Campylobacter infection.     

Investigation into the radiosensitivity of Campylobacter fetus subspecies jejuni in ground chicken meat

Our aim was to study the resistance of Campylobacter fetus subsp. jejuni (CFJ) to irradiation in culture media and in chicken paste as well as to determine whether the radurization doses employed to destro salmonellae were sufficient to kill Campylobacter. It was found that CFJ survived for as long as 16 weeks when stored in culture media at ? 15°C after an irradiation dose of 0.2 kGy. When the dose was increased to 1 kGy, no microbe was detected at all, even if the initial CFJ count had been 10⁹ ml^?1. In chicken paste there was a lesser degree of destruction. After low irradiation doses (0.1, 0.25, 0.5 kGy) the CFJ count decreased in proportion to increasing dosage. When a dose of 1 kGy was given, the initial 10⁸ g^?1 CFJ count was reduced to zero, one week after irradiation. Campylobacter fetus subsp. jejuni did not survive a 1 kGy radiation treatment in culture media, or in chicken paste. Therefore the 3–5 kGy dose generally used for meat products for the destruction of salmonellae will also destroy campylobacters.