The Campylobacter isolates used in this study were isolated during a separate study investigating the efficacy of sanitizers during chicken meat processing (Chousalkar et al., 2019). Briefly, chicken meat carcasses (15 birds from each of eight different broiler production sheds) were collected from two separate processing plants prior to the inside-outside wash step. Bacteria were isolated by massaging chicken carcasses (prior to sanitizer exposure) in buffered peptone water (BPW) (Oxoid, Australia). Two hundred microliters of BPW wash was spread plated onto modified charcoal-cefoperazone deoxycholate agar (mCCDA) (Oxoid, Australia) and incubated at 42°C in 10% CO₂ for 48 h. Putative, Campylobacter isolates were sub-cultured once to obtain pure cultures, stored at −80°C in 5% glycerol, and further characterized using PCR. A total of 116 Campylobacter isolates were obtained.

In preparation for experiments, bacteria were resuscitated from freezing stocks on to Columbia sheep blood agar (SBA) (Oxoid, Thermo Scientific, Australia) and incubated at 42°C in 10% CO₂ for 48 h. The Campylobacter jejuni ATCC 33291 strain was used as a control strain.

Campylobacter isolates were further characterized using a multiplex PCR enabling the distinction of Campylobacter coli (C. coli) and Campylobacter jejuni (C. jejuni) strains. Bacterial DNA was extracted using 0.6% Chelex resin (BioRad, United States) according to the manufacturer’s instructions. Purified DNA was stored at −20°C until required.

The multiplex PCR method designed by Van et al. (2017) enabled the distinction of C. coli and C. jejuni strains. To detect C. jejuni, primers (Forward: 5′-ACT TCT TTA TTG CTT GCT GC-3′, Reverse: 5′-GCC ACA ACA AGT AAA GAA GC-3′) designed to the hipO gene were used. This reaction generated an amplicon of 323 base pairs. To distinguish C. coli, specific primers (Forward: 5′-GCT GCA CTT TTA AAT CCA G-3′, Reverse: 5′-CTT TGG TTT TAC AAT ATG AGC-3′) designed to the glyA gene were used. The C. coli reaction generated an amplicon of 186 base pairs.

The C. coli and C. jejuni multiplex PCR reaction was conducted using a total volume of 20 μL. Each reaction contained 100 ng DNA, 1× My Red Taq reaction buffer, 250 nM each C. jejuni forward and reverse primer, 500 nM each C. coli forward and reverse primer, 0.2 units of My Red Taq polymerase (Bioline, Australia), and nuclease free water. PCR cycling conditions were performed using a Biorad T100 thermocycler. The first step was an initial melt at 98°C for 1 min. The second step included 35 cycles of 98°C for 10 s, 59°C for 30 s, and 72°C for 30 s. A final extension was done at 72°C for 10 min. Products were electrophoresed using a 2% agarose gel.

The chlorine minimum inhibitory concentration (MIC) was determined for both C. jejuni (n = 89) and C. coli (n = 27) isolates. The MIC was determined using the broth microdilution method in nutrient broth number 2 (NB2) (Oxoid, Australia) according to the Clinical and Laboratory Standard Institute guidelines (Weinstein, 2018). Chlorine (4% Sodium hypochlorite, Sigma-Aldrich, Australia) concentrations ranging from 2 ppm to 1024 ppm (pH range 7.5–8.0) were prepared using 96 well round bottom microtiter plates (Thermo Scientific, Australia). Campylobacter inoculums were prepared using a 0.5 McFarland standard and were confirmed measuring the optical density at 600 nm to obtain 10⁸ CFU/mL. Subsequently, 10 μL of the Campylobacter inoculum (10⁶ CFU) was added into 990 μL of chlorine dilutions. The positive control was comprised of NB2 without chlorine but containing bacteria; the negative control was NB2 only. All isolates were tested in duplicate. C. jejuni ATCC 33291 was included as the experimental standard. MIC plates were incubated at 42°C in 10% CO₂ for 20 h. The lowest concentration, which did not give visible bacterial growth was defined as the MIC. Isolates with disparate results in replicate wells were repeated.

After the MIC was determined, wells showing growth inhibition were drop plated on to SBA agar plates to determine the minimum bactericidal concentration (MBC). Briefly, 10 μL of broth from each well in MIC plate was drop plated on to SBA plates and incubated at 42°C in 10% CO₂ for 48 h. The MBC was defined as the lowest bactericidal concentration of chlorine required to kill bacteria after incubation at 42°C in 10% CO₂ for 20 h.

Time-kill assays were used to determine the susceptibility of Campylobacter isolates to chlorine. Two C. jejuni isolates, with disparate MIC values of 128 ppm (C1) and 16 ppm (C2), were selected for this experiment. To determine the effect of organic load, time-kill experiments were performed using both NB2 and 0.9% saline. Standard chlorine concentrations used in the poultry industry ranges between 8–10 ppm when contacting carcasses (Oyarzabal, 2005), hence, dilutions were prepared at half the standard (4 ppm), standard concentration (8 ppm), and twice the standard (16 ppm). The inoculum was prepared by suspending Campylobacter colonies in either NB2 or 0.9% saline and matching the turbidity of an 0.5 McFarland standard. Subsequently, 10 μL of Campylobacter inoculum was added to 990 μL of each chlorine dilution for a final bacterial concentration of 10⁶ CFU/mL and exposed to chlorine for 24 h at either 5°C and 25°C. Bacterial counts were determined at specific intervals post exposure (2, 20, 60, 90, 120, 240, 480, and 1440 min). Serial 10-fold dilutions in 0.9% saline were immediately prepared and 10 μL was drop plated onto SBA and incubated at 42°C in 10% CO₂. Time kill assays were conducted in triplicate and repeated two times.

Microbial resistance to chlorine was evaluated using the chlorine inactivation assay. For these experiments, six C. jejuni isolates were randomly selected based on the MIC and MBC results. Low (0.2–2 ppm) and high (2 ppm–256 ppm) chlorine concentrations were prepared in 0.9% saline. To determine whether bacterial load had an effect on the efficacy of chlorine, two inoculum doses, 10⁸ CFU/mL and 10⁶ CFU/mL, were used. Bacteria were exposed to chlorine for 2 min at 25°C. Bacterial counts were obtained by drop plating 10 μL of serial 10-fold dilutions on to SBA and incubating at 42°C in 10% CO₂ for 48 h. Normal saline without chlorine was used as the bacterial growth control. All isolates were tested in duplicate and each experiment was repeated twice.

After 2 min of exposure to chlorine, 100 μL of all treatment and control groups in the inactivation assay were inoculated into 900 μL of Preston broth (nutrient broth number 2 with Campylobacter selective supplement) (Oxoid, Australia) and incubated at 42°C in 10% CO₂ for up to 48 h. After 24 h and 48 h incubation, 10 μL of each treatment was drop plated on to SBA to determine if the Campylobacter isolate had recovered from exposure to chlorine. Each resuscitation experiment was performed with two replicates and was repeated twice.

Quantifying the amount of UV absorbent material is often used as a measure for bacterial membrane damage (Virto et al., 2005; McKenzie et al., 2016). To characterise chlorine induced membrane damage, 300 μL of each treated and untreated samples were collected after the 2-min exposure (inactivation assay) and centrifuged at 6000 × g for 10 min. UV absorbencies were read at 260 nm and 280 nm with a spectrophotometer (ClarioStar, BMG Lab Tech, Australia). All six isolates were tested in duplicates at 25°C and were repeated.

Electron microscopy was performed to characterise the morphological and cellular changes of C. jejuni isolates after exposure to 8 ppm chlorine for 2 min. After chlorine exposure, the bacterial pellets of both control and treatment was obtained by centrifuging at 8000 × g for 10 min. The pellet was resuspended in normal saline and washed twice to remove excess chlorine. Subsequently, the pelleted cells were fixed in electron microscopy fixative (4% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Sample preparation and microscopy were conducted at Adelaide Microscopy, The University of Adelaide, Adelaide SA, Australia.

The bacterial pellet was washed twice in PBS with 4% sucrose for 10 min, followed by post-fixation with 1% Osmium tetroxide for 1 h. After washing, bacteria were dehydrated in a graded ethanol series (70, 95, and 100%), substituted with propylene oxide, embedded in LX112 epoxy resin, and placed in a 60°C oven for 24 h to polymerize the resin. Ultrathin (60–80 nm) sections were prepared by cutting resin-fixed pellets with an RMC Power Tome Ultramicrotome and double stained with uranyl acetate and lead citrate. Sections were examined utilizing a TEI Tecnai G2 Spirit Bio TWIN at 20–120 kV and imagining was done via in – column Olympus-SIS Veleta CDD camera.

C. jejuni cultures were filtered through 0.2 μm membrane filters (Whatman, United States), washed with PBS + 4% sucrose and fixed with 2% osmium tetroxide for 45 min. After washing, cells were dehydrated in a graded ethanol series (70, 90, and 100%). The filters were then dried with 1:1 ratio of 100% ethanol: hexamethyldilazane for 30 min followed by 100% hexamethyldilazane for 10 min. Cultures were air dried, mounted on carbon stubs, and sputter coated with platinum. Specimens were examined under FEI DualBeam (FIB/SEM) scan electron microscope, operated at 10 kv.

Experimental data for bacterial counts and UV absorbance data are presented as mean ± standard error. One-way Analysis of Variance (ANOVA) and Two-way ANOVA followed by Tukey’s multiple comparison test were used to determine statistical differences of the effects of chlorine on Campylobacter bacterial load. Prevalence data were analyzed using Fisher’s exact test.

All statistical analyses were performed using either SPSS Version 25 (IBM, United States) or GraphPad Prism Version 8.3.0 (GraphPad Software, Inc., United States). In all cases, a P-value of < 0.05 was considered statistically significant.

Putative Campylobacter colonies were collected from positive chicken carcasses and tested using the C. jejuni and C. coli multiplex PCR. A total of 116 isolates were obtained and of these, 76.7% (89/116) were positive for the C. jejuni amplicon while 23.2% (27/116) were positive for the C. coli amplicon. The percentage of C. jejuni isolates collected from the chicken meat samples was significantly higher (P < 0.05) than C. coli.

The bactericidal activity of chlorine was tested over a range of concentrations from 2 to 1028 ppm. The MICs and MBCs obtained for both C. jejuni and C. coli isolates are shown in Figures 1A,B, respectively. The majority of both C. jejuni (57.3%) and C. coli (55.5%) isolates exhibited an MIC of 128 ppm and this result was significant (P < 0.05). MBC values were similar for 59.5% of C. jejuni and 66.6% of C. coli isolates exhibiting an MBC of 128 ppm. No significant difference was observed for MIC or MBC values between Campylobacter species.

Organic material present in the NB2 media used for MIC and MBC tests likely reduced the biocidal effect of chlorine on Campylobacter. To confirm this, time kill curves were performed using both NB2 and 0.9% saline. Two C. jejuni isolates with disparate chlorine MIC values were selected for this experiment. Isolate C1 had an MIC of 128 ppm while the MIC for C2 was 16 ppm.

Data are presented as mean log₁₀ CFU/mL C. jejuni. At 5°C, no significant effect of chlorine was observed on the total C. jejuni load over time in NB2 (Figure 2A). At 25°C in NB2, the total bacterial load did not vary significantly between 2 min and 8 h of exposure at any of the chlorine concentrations tested (Figure 2B). A significant reduction (P ≤ 0.001) in bacterial loads was observed at 25°C for all treatment groups after 24 h of exposure. A 3-log reduction was observed at 24 h for the C1 treatment groups while a 4-log reduction was observed all C2 treatment groups. Interestingly, the reduction in bacteria observed at 25°C after 24 h of did not occur at 5°C.

In 0.9% saline, the culturability of both C. jejuni isolates C1 and C2 was completely inhibited following exposure to chlorine. After 2 min of exposure, no culturable bacteria (either C1 or C2) were obtained from all three chlorine concentrations at either 5 or 25°C (Figures 2C,D). No significant difference in the mean bacterial load was observed for the control groups between 2 min and 8 h. After 24 h, however, culturable bacteria were not detected at either temperature.

Based on results from the time kill experiments, two minute inactivation experiments were conducted for the six C. jejuni isolates using low (0.2 – 2.0 ppm) and high (2 – 256 ppm) concentrations of chlorine (Figure 3). At 10⁶ CFU/mL, all six isolates were highly sensitive to chlorine at both low and high concentrations and were not culturable following a 2-min exposure (Figures 3A,B). Interestingly, at this load, none of the C. jejuni isolates were cultured from the lowest chlorine concentration of 0.2 ppm (Figure 3A).

At 10⁸ CFU/mL, a significant effect of chlorine was observed for both the low (P < 0.001) and high (P < 0.001) range of concentrations tested. C3, and C4 were the most sensitive C. jejuni isolates. At 1.8 and 2.0 ppm of chlorine, culturable C3 bacteria were no longer detected while C4 exhibited a 3 log₁₀ reduction in the total number of culturable bacteria. C3, and C4 exhibited significantly lower (P < 0.001) bacterial loads at chlorine concentrations between 1.4–2.0 ppm compared with C2, C5, and C6 isolates. At the low range of chlorine concentration, no significant change in the number of culturable C6 was observed.

A significant effect of chlorine was observed over the high concentration range (P < 0.001). Consistent with the low range data, isolate C3 was not culturable at 2 ppm or higher. Significant variability was observed amongst the isolates in their response to chlorine (P < 0.001). Isolate C6 retained the highest bacterial loads up to 8 ppm where it was no longer culturable. C1 and C5 remained culturable at 8 ppm but there was no significant difference between the bacterial loads. No isolate was cultured from chlorine suspensions ranging from 16–256 ppm.

After 2 min of exposure to different chlorine concentrations, 100 μL of each Campylobacter isolate were added to resuscitation media to determine sub-lethal injury. The number of non-culturable isolates and whether they were resuscitated is shown in Table 1. Non-culturable Campylobacter from the 10⁶ CFU/mL chlorine treatment groups were not resuscitated after either 24 or 48-h incubation in Preston broth.

Bacterial resuscitation in Preston broth was also performed for 10⁸ CFU/mL C. jejuni treatment groups following exposure to chlorine. At 2 ppm, 1/6 isolate was non-culturable but was resuscitated after both 24 and 48-h incubation in broth. Similarly, at 4 ppm, 2/6 isolates were non-culturable following chlorine exposure and were both resuscitated. All C. jejuni isolates were resuscitated in broth after exposure to 8 ppm chlorine. After exposure to both 32 and 64 ppm chlorine, 6/6 isolates were non-culturable but only one isolate was resuscitated at both concentrations. It should be noted that this was the same isolate. None of the C. jejuni isolates were culturable after exposure to 128 or 256 ppm and could not be resuscitated.

The values of UV absorbance at 280 nm of the supernatants collected from 10⁶ to 10⁸ CFU/mL C. jejuni suspensions after exposure to low and high chlorine concentrations are shown in Figure 4. At 10⁶ CFU/mL, membrane damage following exposure to low concentrations of chlorine (0.2–2 ppm) was minimal as UV absorbance values observed were less than 0.150 (Figure 4A) and no significant effect of chlorine was observed. Increasing concentration of chlorine resulted in significantly higher (P < 0.001) 280 nm absorbance values for the 10⁶ CFU/mL C. jejuni suspension (Figure 4B).

Higher absorbencies were observed for the 10⁸ CFU/mL C. jejuni suspension over the low concentration range as compared with the 10⁶ CFU/mL suspension (Figure 4C). At low chlorine concentrations, a significant increase in absorbance at 280 nm was observed for the10⁸ CFU/mL suspension (P < 0.001). Similar results were observed for the high chlorine range. A significant effect of concentration (P < 0.01) was detected for the 10⁸ CFU/mL suspensions with increasing UV absorbencies observed between 2 – 256 ppm (Figure 4D). Variation in UV absorbance values were observed between strains especially when the bacterial load was high but no significant difference between isolates was detected. Of note, isolate C3, which was inactivated at the lowest concentrations exhibited the highest absorbance values at both low (Figure 4B) and high (Figure 4D) bacterial concentrations. The results obtained at 260 nm (data not shown) were similar to those obtained at 280 nm.

Based on the UV absorbance data, we investigated the morphological changes in C. jejuni after exposure to chlorine using SEM (Figure 5) and TEM (Figure 6). The cell structure of C. jejuni following a 2-min exposure to 8 ppm of chlorine was substantially altered. Under SEM, C. jejuni cells appeared either coccoid or exhibited a stretched and shriveled morphology (Figures 5B–D). Loss of integrity and complete destruction of the cell membrane was also observed (Figure 5B). Unaffected cells exhibiting normal C. jejuni morphology were also observed in the chlorine treatment group (Figure 5D, white arrow). TEM analysis of C. jejuni exposed to chlorine revealed that treated cells were not dense as cells in the control (Figures 6D–F). The bacterial cells in various stages of degeneration were also observed. The cytoplasm of some bacterial cells was detached from the cell membrane (Figure 6D, white arrow). Cells with severely damaged the cell membranes and extruded cytoplasm were also frequently observed (Figures 6D,E).