Research Note: Detection of Campylobacter spp. in chicken meat using culture methods and quantitative PCR with propidium monoazide

Globally, Campylobacter spp. are prominent causative agents of food-borne gastroenteritis. These pathogens are commonly detected using conventional culture methods; however, culture methods are unable to detect viable but nonculturable (VBNC) bacteria. Currently, the detection rate of Campylobacter spp. on chicken meat does not correlate with the seasonal peak of human campylobacteriosis. We hypothesized that this may be due to the presence of undetectable VBNC Campylobacter spp. Therefore, we previously established a quantitative PCR assay using propidium monoazide (PMA-qPCR), which can detect viable Campylobacter cells. In this study, PMA-qPCR was conducted to detect viable Campylobacter spp. in chicken meat, and the detection rates of PMA-qPCR and the culture method throughout all 4 seasons were compared. A total of 105 chicken meat samples (whole legs, breast fillets, and livers) were screened for the presence of Campylobacter spp. using both PMA-qPCR and the conventional culture method. The detection rates of the 2 methods did not differ significantly; however, the positive and negative samples were not always consistent. Detection rates in March were significantly lower compared to months with the highest detection rates. These results suggest that, to increase the detection rate of Campylobacter spp., the 2 methods should be used in parallel. In this study, PMA-qPCR could not detect VBNC Campylobacter spp. effectively in C. jejuni-spiked chicken meat. Further studies using improved viability-qPCR should be performed to describe the impact of the VBNC state of Campylobacter spp. on the detection of this bacterium in chicken meat.


INTRODUCTION
Campylobacter spp. are the leading cause of foodborne bacterial gastroenteritis worldwide and is mostly associated with contaminated chicken meat (Skirrow, 1977). According to statistics from the Japanese Ministry of Health, Labour, and Welfare, Campylobacter jejuni and Campylobacter coli have been the predominant cause of bacterial food poisoning in Japan since 2003 (https://www.mhlw. go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/ syokuchu/04.html [accessed on March 14, 2023]).
Globally, conventional culture methods are commonly used to detect Campylobacter spp. in poultry. However, exposure to various environmental stressors, such as low temperature, nutrient starvation, and unsuitable aerobic conditions can cause Campylobacter spp. to enter a viable but nonculturable (VBNC) state (Yagi et al., 2022). PCR or quantitative PCR (qPCR) are useful alternatives to conventional culture methods for the detection of both culturable and VBNC organisms. However, both PCR and qPCR can amplify bacterial DNA originating from nonviable cells that will not cause food poisoning. To solve this problem, propidium monoazide (PMA), which only enters dead bacteria through the damaged cell wall/cell membrane, binds to the DNA, and prevents DNA amplification via PCR, has been combined with PCR or qPCR to detect only viable bacteria .
The detection rate of Campylobacter spp. in poultry varies by season, increasing in warmer seasons and dropping during the winter (Meldrum et al., 2005). However, the number of human campylobacteriosis case in Japan reaches a maximum in June, preceding the increase in Campylobacter-contaminated chicken meat samples observed from June to November (Ishihara et al., 2012). Therefore, the variation in the amount of Campylobacter spp. detected in chicken meat is suggested to not be the primary reason for the seasonality of human campylobacteriosis (Meldrum et al., 2005, Williams et al., 2015. We hypothesized that the low detection rate in winter might be due to the presence of VBNC Campylobacter spp. that remain undetected by conventional culturing methods. Previously, we optimized a protocol for PMA treatment followed by qPCR (PMA-qPCR) to distinguish between dead and living cells . Our results suggested that the optimized PMA-qPCR was able to detect VBNC Campylobacter spp. in chicken meat; however, the previous experiment was only conducted for one month. Therefore, in this study, we aimed to compare PMA-qPCR with conventional culture methods and to examine the ability of PMA-qPCR to detect the VBNC state of Campylobacter spp. in chicken meat throughout the year.

Chicken Meat
A total of 105 chicken meat packs were purchased from 4 local supermarkets in the Gifu Prefecture, Japan, from September 2021 to June 2022. The following samples were collected: whole legs (n = 36), breast fillets (n = 36), and livers (n = 33). Samples were transported to the laboratory in isothermal boxes and bacterial isolation was performed within 1 h after purchase.

PMA-qPCR
To perform PMA-qPCR, 25 g of each chicken sample was rinsed with 10 mL of sterilized PBS. The rinsed solution was centrifuged at 800 £ g for 10 min at 4°C to eliminate larger debris. The supernatant was then centrifuged at 8,000 £ g for 10 min at 4°C to pellet bacteria. Cells were subjected to a 2-round PMA treatment, as described previously . Briefly, each pellet was resuspended in 160 mL of PBS, followed by the addition of 40 mL of PMA Enhancer for gram-negative Bacteria (5X solution; Biotium Inc., Fremont, CA; final concentration: 1X) and 0.5 mL of PMAxx solution (20 mM in H 2 O; Biotium Inc.; final concentration: 25 mM). Each sample was thoroughly mixed and incubated in the dark at 37°C for 10 min, followed by light exposure for 15 min using an LED Crosslinker 12 (Takara, Kusatsu, Japan) to activate the PMAxx. Treated cells were centrifuged at 8,000 £ g for 10 min at 4°C and the supernatant was removed. A second round of PMA treatment was performed as described above. Cells were collected using centrifugation at 8,000 £ g for 10 min at 4°C, and DNA was extracted from each pellet using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted DNA was subjected to qPCR using a StepOnePlus thermal cycler (Applied Biosystems, Foster City, CA). To detect Campylobacter spp., specific primers, campF2 and campR2, and a probe, camp2probe, were used (Lund et al., 2004). The PCR mixture was prepared to a final volume of 20 mL, which contained 2 mL of the extracted DNA, using the GoTaq Probe qPCR Master Mix (Promega, Madison, WI), according to the manufacturer's instructions. Thermal cycling was performed under the following conditions: 2 min at 95 C, followed by 40 cycles of 95 C for 3 s and 60 C for 30 s. Fluorescence was measured at the end of each cycle.

Culture-Based Enumeration
The standard method for detection of Campylobacter spp. in Japan, following the NIHSJ-02 guideline, was used (Momose et al., 2013). Approximately 25 g of each chicken sample was added to 100 mL of Preston selective broth comprised of nutrient broth No. 2 (Oxoid, Hampshire, UK), 5% of sterile lysed horse blood, Campylobacter growth supplement (Oxoid), and modified Preston Campylobacter selective supplement (Oxoid). Each sample was mixed for 1 min using a stomacher and then incubated at 42°C for 24 h under microaerobic conditions. Enriched cultures were streaked on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) and Skirrow agar comprised of blood agar base No. 2 (Oxoid), 7% of sterile lysed horse blood, Skirrow Campylobacter selective supplement (Oxoid), and Campylobacter growth supplement. Streaked cultures were incubated at 42°C for 48 h under microaerobic conditions. A colony resembling Campylobacter spp. was selected from each of the samples and identified using colony PCR with C412F and C1228R primers (Linton et al., 1996). Amplicons were purified using ethanol precipitation and sequenced using the C412F and C1228R primers in an ABI 310 Genetic Analyzer with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were subjected to BLAST verification using the NCBI database.

Campylobacter Jejuni-Spiked Chicken Meat
Campylobacter jejuni NCTC11168 strain was purchased from the American Type Culture Collection (ATCC 700819). To generate culturable C. jejuni, cells from the -80°C stock were cultured on mCCDA at 37°C for 2 d under microaerobic conditions. Colonies were suspended in 10 mL of Brucella broth and cultured under microaerobic conditions at 37°C for 36 h at 130 rpm using an incubator shaker (Innova 4300, New Brunswick Scientific, Enfield, CT). C. jejuni cells were resuspended in PBS and diluted to an optical density at 600 nm (OD 600 ) of 1.0 using a spectrophotometer (GeneQuant 100, GE Healthcare, Chicago, IL), which corresponded to approximately 1£10 8 CFU/mL. To generate VBNC C. jejuni, culturable cells were resuspended in Muller-Hinton (MH) broth (275730, BD Biosciences, Billerica, MA), adjusted to 1 £ 10 8 CFU /mL, and cultured at 4°C for 30 d under aerobic conditions as previously described (Yagi et al., 2022). Cell viability was examined using a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Carlsbad, CA).
Liquid cultures from culturable and VBNC cells were adjusted to 1 £ 10 6 CFU/mL via serial dilution of the stock solution (1 £ 10 8 CFU/mL). To generate the C. jejuni-spiked chicken meat, 100 mL of 1 £ 10 6 CFU/mL bacterial suspension was applied to 25 g of chicken meats (whole legs). The meat was confirmed a priori to be Campylobacter spp. free using both culture method and PMA-qPCR. C. jejuni-spiked chicken meat was put in the refrigerator overnight to simulate Campylobacter spp. contaminated chicken meat. Culturable and VBNC C. jejuni were detected via qPCR with and without PMA treatment.

Statistical Analysis
Statistical analysis of results was performed using McNemar's and chi-square tests (Bonferroni correction). Results with P-values < 0.05 were considered significant.

RESULTS AND DISCUSSION
We compared PMA-qPCR with culture methods for the detection of Campylobacter spp. in chicken meat. The prevalence of Campylobacter spp. in commercial chicken meat as detected by both methods is summarized in Table 1. No significant difference was found between the detection rate of PMA-qPCR and that of the culture method for whole legs, breast fillets, and livers. The number of positive and negative results determined using both methods is summarized in a 2 £ 2 matrix ( Table 2). The results obtained using the 2 methods were not consistent.
In samples that tested positive using PMA-qPCR, but negative using the culture method, we predicted that Campylobacter spp. were in a VBNC state. To test whether PMA-qPCR could effectively detect VBNC Campylobacter spp., we performed experiments using culturable and VBNC C. jejuni-spiked chicken meat. The viability of C. jejuni was confirmed to be more than 80% in both culturable and VBNC cells. VBNC cells were confirmed not to form colonies on mCCDA. Culturable and VBNC C. jejuni cells were effectively detect via qPCR without PMA treatment (Ct values ranging from 21 to 23). Conversely, using PMA-qPCR, VBNC state of C. jejuni was not effectively detect, presenting Ct values above 35 or ''not detected'', whereas culturable C. jejuni was effectively detected (Ct values ranging from 23 to 24). These results indicated that PMA-qPCR not always detected VBNC cells. Therefore, there might be 2 reasons for Campylobacter spp. not being detected using the culture method but detected via PMA-qPCR. First, Campylobacter spp. were in VBNC state. Second, the growth of Campylobacter spp. in culture media was affected by some substances present in chicken meat. To detect VBNC state of C. jejuni effectively, an improved viability-qPCR should be established.
Samples that were positive using both methods could have contained bacteria in both a culturable and VBNC state. In PMA-qPCR-negative but culture-positive samples, a relatively small number of Campylobacter spp. were detected using the culture method, which could be attributed to the use of enriched media. As the number of Campylobacter spp. in the chicken meat was not counted, quantitative tests should be performed to confirm this hypothesis. To increase the detection rate of Campylobacter spp., the 2 methods may be used in parallel (Table 2). However, as C. jejuni doses of more than 800 CFU is required for human infection (Black et al., 1988), detecting a small number of bacteria is not feasible, which can only be detected using enrichment culture methods.
In conclusion, this study demonstrated that there is no difference between the detections rates of Campylobacter spp. in chicken meat using PMA-qPCR and the culture method. Furthermore, our results indicate that the lower detection rate of Campylobacter spp. occurs in winter season using culture method and PMA-qPCR. However, PMA-qPCR could not detect VBNC Campylobacter spp. effectively. Further studies using improved viability-qPCR should be performed to describe the impact of the VBNC state of Campylobacter spp. on the detection of this bacterium in chicken meat.