Antimicrobial Resistance Gene Transfer from Campylobacter jejuni in Mono- and Dual-Species Biofilms

ABSTRACT

Horizontal gene transfer (HGT) is a driving force for the dissemination of antimicrobial resistance (AMR) genes among Campylobacter jejuni organisms, a leading cause of foodborne gastroenteritis worldwide. Although HGT is well documented for C. jejuni planktonic cells, the role of C. jejuni biofilms in AMR spread that likely occurs in the environment is poorly understood. Here, we developed a cocultivation model to investigate the HGT of chromosomally encoded AMR genes between two C. jejuni F38011 AMR mutants in biofilms. Compared to planktonic cells, C. jejuni biofilms significantly promoted HGT (P < 0.05), resulting in an increase of HGT frequencies by up to 17.5-fold. Dynamic study revealed that HGT in biofilms increased at the early stage (i.e., from 24 h to 48 h) and remained stable during 48 to 72 h. Biofilms continuously released the HGT mutants into supernatant culture, indicating spontaneous dissemination of AMR to broader niches. DNase I treatment confirmed the role of natural transformation in genetic exchange. HGT was not associated with biofilm biomass, cell density, or bacterial metabolic activity, whereas the presence of extracellular DNA was negatively correlated with the altered HGT frequencies. HGT in biofilms also had a strain-to-strain variation. A synergistic HGT effect was observed between C. jejuni with different genomic backgrounds (i.e., C. jejuni NCTC 11168 chloramphenicol-resistant strain and F38011 kanamycin-resistant strain). C. jejuni performed HGT at the frequency of 10⁻⁷ in Escherichia coli-C. jejuni biofilms, while HGT was not detectable in Salmonella enterica-C. jejuni biofilms.

IMPORTANCE Antimicrobial-resistant C. jejuni has been listed as a high priority of public health concern worldwide. To tackle the rapid evolution of AMR in C. jejuni, it is of great importance to understand the extent and characteristics of HGT in C. jejuni biofilms, which serve as the main survival strategy of this microbe in the farm-to-table continuum. In this study, we demonstrated that biofilms significantly enhanced HGT compared to the planktonic state (P < 0.05). Biofilm cultivation time and extracellular DNA (eDNA) amount were related to varied HGT frequencies. C. jejuni could spread AMR genes in both monospecies and dual-species biofilms, mimicking the survival mode of C. jejuni in food chains. These findings indicated that the risk and extent of AMR transmission among C. jejuni organisms have been underestimated, as previous HGT studies mainly focused on the planktonic state. Future AMR controlling measures can target biofilms and their main component eDNA.

INTRODUCTION

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis worldwide. Campylobacter spp. are estimated to account for 400 to 500 million cases of diarrhea per year, among which C. jejuni contributes to ∼90% of infections (1, 2). As a foodborne pathogen, C. jejuni prevalently colonizes the intestinal tracts of food-producing animals and is frequently exposed to antibiotics in animal husbandry settings, resulting in resistance against clinically important antibiotics. The antimicrobial resistance (AMR) of C. jejuni emerges through either horizontal gene transfer (HGT) for the acquisition of AMR genes or spontaneous point mutation on chromosomes for modifying antibiotic target sites (3). Compared to spontaneous mutation, HGT of large DNA segments can introduce a larger number of polymorphisms and generate novel phenotypes more rapidly (2). This high efficiency of HGT was evident in the spread of AMR genes between Campylobacter spp. (4–6) or from Gram-positive bacteria to Campylobacter (7, 8). A recent survey study reported that 90.4% of Campylobacter isolates recovered from food animals, poultry processing facilities, and retail meat in North Carolina carried at least one AMR gene, while 43% obtained AMR genes for three or more antibiotic classes (9). About 50% of Campylobacter isolates in broiler chicken in Canada were found resistant to at least one antibiotic class (10). Of particular concern, the World Health Organization has marked the AMR crisis of Campylobacter as a high priority to resolve (11).

Natural transformation is a well-recognized HGT mechanism in C. jejuni by which bacterial cells take up free DNA (i.e., plasmids and genomic DNA [gDNA]) from the environment. Bacteria either maintain the plasmids or integrate gDNA into chromosomes through homogenous recombination (3, 12–14), obtaining new genotypic and/or phenotypic traits. The natural transformation of C. jejuni was first reported by Wang and Taylor in 1990, demonstrating the transformation frequencies varying from <10⁻⁶ to 10⁻⁴ (14). Since then, the mechanisms and characteristics of natural transformation in C. jejuni have been extensively investigated using planktonic culture as a standard model (14–16). Many factors influence the transformation frequency in C. jejuni planktonic cells, including bacterial growth phases, cell density, source of DNA, strain type, and atmosphere (14–16). Despite the fact that C. jejuni in the planktonic state can be cultivated under well-controlled laboratory conditions, they are sensitive to environmental and food processing-related stressors, such as oxygen atmosphere and fluctuating temperatures (17). As a result, the HGT studies associated with planktonic cells may not be a good predictor for the rapid evolution of AMR in C. jejuni along food chains.

Biofilms likely represent one of the primary survival strategies for C. jejuni in the farm-to-table continuum, embedding the bacteria by their self-produced polymeric matrix and protecting them from various stressors (17). C. jejuni not only forms mono-species biofilms but has been recovered from the mixed-species biofilms associated with chicken houses (18), poultry-processing plants (19), and water systems (20). Biofilm sessile cells have increased resistance to antimicrobials compared to planktonic cells, attributed to the reduced diffusion of antimicrobials through biofilm matrix, production of enzymes that degrade antimicrobials, and physiological changes of bacteria (21). Moreover, biofilms are known to promote HGT of AMR genes in many Gram-positive and Gram-negative bacteria, such as Streptococcus spp. (22, 23), Pseudomonas aeruginosa (24), and Vibrio cholerae (25). The presence of extracellular DNA (eDNA) is concentrated in the biofilm matrix, which stabilizes biofilm structure and provides a genetic pool for HGT (26). Our group has recently demonstrated that C. jejuni employs a bacterial lysis mechanism to release eDNA so as to mediate biofilm formation (27). The release of eDNA from the C. jejuni biofilms is expected to provide the source of AMR genes for natural transformation. However, few studies have investigated the natural transformation of AMR genes in C. jejuni biofilms (28, 29); thus, the critical parameters influencing the efficiency of HGT within a biofilm are not known.

In this study, we developed a cocultivation model to systematically assess the role of biofilm and its intrinsic characteristics on C. jejuni HGT. Two representative AMR genes, namely, aphA-3 (encoding kanamycin resistance) and cat (encoding chloramphenicol resistance), were separately integrated into the chromosome of C. jejuni as the markers of genetic exchange. Our study aimed to answer four questions. (i) How do different factors influence HGT frequencies, such as the modes of growth (i.e., planktonic cells, biofilm sessile cells, and biofilm supernatant cells), incubation time, and strain variation? (ii) What is the main mechanism of the emergence of dual resistance mutants in C. jejuni biofilms, either through natural transformation or spontaneous mutation? (iii) Is there any correlation between biofilm characteristics and natural transformation? (iv) How does C. jejuni perform natural transformation in dual-species biofilms? Understanding the HGT in C. jejuni biofilms can lead to broader insights into the ability of this pathogen to obtain AMR and adapt to the environment.

RESULTS

Construction and characterization of C. jejuni AMR marker strains.

We selected C. jejuni F38011 as a model strain for the HGT study, given that it produces a higher level of biofilm than many C. jejuni reference strains under optimal laboratory growth condition (27), and its whole-genome sequence is available for genetic manipulation (NCBI accession no. NZ_CP006851.1). To monitor the potential genetic exchange between C. jejuni strains in biofilms, C. jejuni F38011 was engineered by adding either the aphA-3 or cat gene into the bacterial chromosome to provide resistance to kanamycin (Kan^r) or chloramphenicol (Cm^r), respectively. Previous HGT studies commonly inserted the AMR genetic markers into genes that encode nonessential enzymes or that do not interrupt relevant performances such as host colonization (16, 30). However, the disruption of certain genes may still influence bacterial physiology. Instead, we introduced the Kan^r or Cm^r cassette into the chromosomal noncoding region between 23S rRNA and 5S rRNA or the one between 16S rRNA and tRNA-Ala, respectively (see Fig. S1A in the supplemental material). The noncoding regions of the rRNA gene cluster were selected because there are three highly conserved copies of the ribosomal gene cluster, potentially minimizing the effects of AMR gene addition on the function of rRNA.

In this study, Kan^r and Cm^r genes were successfully inserted into the defined genomic loci of C. jejuni F38011 through homogenous recombination, as confirmed by PCR (data not shown). To assess if AMR genes were properly expressed in the mutants, we conducted the broth microdilution method for the determination of MIC values. Compared to the C. jejuni F38011 parental strain, the Kan^r and Cm^r mutants showed a decreased susceptibility to kanamycin and chloramphenicol, respectively (Table S1). Besides, the presence of resistance genes in rRNA gene clusters did not influence the bacterial growth rate (Fig. S1B) or biofilm formation (Fig. S1C), indicating no significant (P > 0.05) fitness cost was introduced in the current study model. Therefore, we used these two C. jejuni F38011 AMR mutants in the following HGT studies.

HGT was dependent on bacterial growth mode and incubation time.

HGT between C. jejuni strains has been reported in liquid shake culture (15) and chickens (30), contributing to bacterial genetic diversity and AMR evolution. We hypothesized that HGT between C. jejuni F38011 isogenic AMR strains could also occur in the biofilm state. To test this hypothesis, equal concentrations of C. jejuni F38011 Kan^r and F38011 Cm^r isolates were mixed and inoculated into a polystyrene 24-well plate. The plate was statically incubated for 24 to 72 h under an optimal growth condition (i.e., 37°C, microaerobic, Muller-Hinton [MH] broth). To monitor the emergence of HGT, biofilm sessile cells were enzymatically removed from the 24-well plate using 1% trypsin-EDTA solution and subsequently spread onto MH agar supplemented with 5% defibrinated sheep blood (MHBA), chloramphenicol (20 μg/ml), and kanamycin (50 μg/ml). Prior to harvesting the sessile cells, the supernatant in the 24-well plates was also enumerated, reflecting the mixture of newly developed AMR mutants in the planktonic state and one released from biofilms to environmental niches. In parallel, the cocultures of Kan^r and Cm^r isolates were incubated in shaking glass tubes to represent the planktonic state.

Regardless of the time, a significantly higher frequency of dual-resistance (Kan^r + Cm^r) mutants was observed in biofilms than that of the planktonic state (P < 0.05), with an increase by 6.4- to 17.5-fold (Fig. 1). The dual-resistance mutants were randomly selected for PCR, and all tested isolates acquired both aphA-3 and cat genes, confirming the role of HGT on AMR evolution (data not shown). In biofilms, Kan^r Cm^r dual-resistance mutants appeared soon after 24 h postinoculation (the first sampling time) at a frequency of 2.71 × 10⁻⁸ per total cells and increased to the maximal and stable frequencies during 48 to 72 h (i.e., ∼4 × 10⁻⁷) (Fig. 1). In contrast, no detectable HGT mutants were identified in the planktonic population at 24 h, as shown in Fig. 1. The HGT frequency of shaking liquid culture peaked at 72 h with a value of 7.01 × 10⁻⁸, which might be contributed by both free-floating planktonic cells and visible biofilm flocs in the glass tube. Taken together, biofilm sessile cells of C. jejuni F38011 performed HGT more frequently and rapidly than their planktonic counterparts.

FIG 1.

Horizontal gene transfer (HGT) frequency of C. jejuni F38011 Kan^r and Cm^r strains under optimal growth condition (37°C, Difco MHB, microaerobic conditions). One-way ANOVA followed by Games-Howell test was conducted to determine the significant differences between different modes of growth (*, P < 0.05; **, P < 0.01). Means and standard deviations (i.e., error bars) were calculated from six independent experiments. LOD, limit of detection.

Biofilms readily released the newly developed AMR mutants into the environment.

In addition to the increased efficiency of HGT within the biofilms, we also identified a prominent number of Kan^r Cm^r dual-resistance mutants in the growth medium of the same system, with the HGT frequencies increasing from 9.45 × 10⁻⁹ to 1.28 × 10⁻⁶ as a function of incubation time (Fig. 1). The presence of dual-resistance mutants in the biofilm supernatant might not solely rely on gene transfer between supernatant cells, since the HGT frequencies in the biofilm supernatant were significantly higher (P < 0.01) than the ones in planktonic cells (Fig. 1), even though both were regarded as a free-floating planktonic state. C. jejuni biofilms have been previously reported to liberate relatively high numbers (∼10⁶ CFU/ml) of viable cells into the environment (31). This spontaneous releasing model was in line with our results in Fig. 1, where HGT frequencies in the biofilm supernatant increased simultaneously along with biofilm sessile cells.

The presence of external flow forces could facilitate the spontaneous release of biofilm cells. To address this possibility, we replaced the growth medium every 24 h to mimic the recurrence of food juice on contact surfaces, serving as nutrients for biofilm formation and mechanical forces for biofilm dispersal. With the addition of fresh MHB, C. jejuni single- and dual-resistance mutants in the biofilms were readily released into the solution (Fig. S2), generating new populations of planktonic cells. Once entering the planktonic state, dual-resistance C. jejuni F38011 had growth rates similar to that of the single-resistance strains (Fig. S3). With the presence of mechanical forces (i.e., adding fresh MHB), the proportion of released HGT mutants in the total population was negatively associated with biofilm age. When biofilms were 24 h old, the number of HGT mutants that were released into the fresh MHB was 7.1 times more than that shed in the biofilms (Fig. S2). This explained the phenomena that the HGT frequency of biofilm supernatant cells was significantly higher (P < 0.05) than that of biofilm sessile cells after an extra 24 h of incubation (i.e., total of 48 h) (Fig. 1). The proportion of released HGT mutants was highly diverse at the early biofilm formation stage (i.e., 24 h) (Fig. S2), which might be due to the uneven distribution of limited HGT mutants in the biofilms. While biofilms were grown for over 48 h, an equivalent amount of dual-resistance mutants was obtained in the fresh MHB and biofilms (Fig. S2), leading to comparable HGT frequencies identified in the supernatant and biofilms at 72 h (Fig. 1). Overall, C. jejuni biofilms could liberate AMR mutants to the environment either spontaneously or under mechanical forces.

Natural transformation played a major role in chromosomal genetic exchange in C. jejuni biofilms.

The evolution of AMR isolates may be caused by spontaneous mutation and/or HGT (32). In this study, we first investigated whether spontaneous mutation contributed to the presence of Kan^r Cm^r dual-resistance mutants in C. jejuni F38011 biofilms. Either C. jejuni F38011 Kan^r or Cm^r biofilms were separately cultivated in 24-well plates for 72 h. No dual-resistance mutants were recovered from biofilm cells after streaking onto MHBA supplemented with 50 μg/ml kanamycin and 20 μg/ml chloramphenicol (data not shown). It indicated that spontaneous mutation was negligible in our experimental model. Next, we carried out a DNase I assay to assess the role of natural transformation on the acquisition of dual-resistance profiles. C. jejuni is generally regarded as natural competence that can take up eDNA from the environment and integrate it into their chromosomes (14). Addition of DNase in biofilm growth medium could reduce the availability of eDNA, thereby decreasing the efficiency of natural transformation. In this study, we incubated the coculture of C. jejuni F38011 Kan^r and Cm^r strains in 24-well plates for 48 h, with the presence of 0 or 100 U/ml DNase I. As shown in Fig. 2, HGT frequency significantly declined in the DNase I-treated biofilms (P < 0.001), while total sessile cell numbers were not affected. Besides, the genome of C. jejuni F38011 lacks plasmids and prophages, ruling out the possibility of conjugation (33) and transduction (34) in the evolution of AMR. In conclusion, natural transformation was the driving force in the spread of AMR genes in C. jejuni F38011 biofilms under the current laboratory settings.

FIG 2.

DNase I treatment for C. jejuni F38011 Kan^r + Cm^r biofilms at 48 h. The horizontal gene transfer (HGT) frequency (A) and biofilm sessile cell counts (B) were quantified by the plating assay. Student's t test was performed for statistical analysis (NS indicates P > 0.05). Means and standard deviations (i.e., error bars) were calculated from six independent experiments.

Relevance between biofilm characteristics and gene transfer rates.

To understand the interconnection between biofilms and HGT, we characterized the temporal profiles of biofilm properties within 72 h and correlated them to HGT rates. The target biofilm properties included cell density, metabolic activity, biofilm biomass, and eDNA amounts (Fig. 3). For C. jejuni planktonic cells, the levels of transformation were previously identified to arise at increased cell density when cocultivating two C. jejuni 81-176 AMR marker strains in shaking liquid (15). To assess if cell density of biofilms was correlated with HGT frequency, we quantified the total cell population in C. jejuni F38011 Kan^r/Cm^r biofilms by the plating assay. Figure 3A showed that the total cell counts in biofilms kept constant during the entire incubation period (24 to 72 h), whereas HGT frequencies increased from 24 h to 48 h (Fig. 1). In other words, cell density in C. jejuni biofilms could not explain the change of HGT efficiency over time.

FIG 3.

Characterization of biofilms during horizontal gene transfer (HGT). (A) Total biofilm cell numbers were quantified by the plating assay. (B) 2,3,5-Tryphenyl tetrazolium chloride (TTC) assay was carried out to determine the metabolic activity of biofilm cells. (C) Crystal violet staining assay was used to determine biofilm biomass. (D) Extracellular DNA (eDNA) within biofilms was quantified by fluorescent SYBR green I dye at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. (E) Pearson correlation analysis was performed to assess the correlation between HGT frequency and biofilm eDNA concentration. One-way ANOVA followed by Tukey’s test was used to determine the statistical difference among different incubation times (*, P < 0.05; NS, no significance).

A previous study reported that the natural transformation of C. jejuni NCTC 11168 at the planktonic state was an energy-dependent process (35). To test if this is a case for C. jejuni biofilms, we determined the metabolic activity of biofilm sessile cells using 2,3,5-triphenyl-tetrazolium chloride (TTC) assay. TTC is one of the most widely used reagents in biology for measuring the metabolic activity of cells. When cells are metabolically active, they can convert colorless TTC solution to a deep red formazan derivative that can be easily quantified by colorimetric-based spectrometry (36). In this study, no significant difference (P > 0.05) was observed between the absorbance values of the TTC assay at different incubation time periods (Fig. 3B). This result did not follow the same trend of HGT frequencies at the corresponding time points (Fig. 1), suggesting the lack of correlation between metabolic activity and HGT in C. jejuni biofilms.

Besides sessile cells, biofilms also consist of extracellular polymeric substances (EPS) that are responsible for the three-dimensional architecture of biofilms (37). EPS keep the embedded biofilm cells close to each other, allowing for intense interactions, such as cell-cell communication and gene transfer (38, 39). In most biofilms, EPS account for over 90% of the dry mass, while sessile cells only occupy less than 10% (39). To explore the relevance between biofilm biomass (i.e., a sum of EPS and cells) and HGT, we performed a crystal violet staining assay on C. jejuni F38011 Kan^r/Cm^r cocultured biofilms. The crystal violet dyes unspecifically bind to the molecules with negative charges that are found on both biofilm sessile cells and EPS (40). According to Fig. 3C, the quantity of biofilm biomass was unchanged from 24 h to 72 h and, thus, did not show a correlation with increased HGT frequencies over time.

We identified a negative correlation between eDNA concentrations and HGT frequency in C. jejuni F38011 Kan^r/Cm^r cocultured biofilms, with a Pearson correlation coefficient of −0.669 and a P value of 0.011 (Fig. 3D and E). The eDNA concentration was about 2-fold higher in the 24-h-old biofilms than that during 48 to 72 h (Fig. 3D), whereas the HGT frequencies at 48 to 72 h were significantly higher (P < 0.05) than that at 24 h (Fig. 1). This phenomenon was in agreement with the observations of the first Campylobacter natural transformation study (14). Each naturally competent Campylobacer cell could probably take up an average of two DNA molecules from the environment (14). We speculated that the 2-fold reduction of eDNA in biofilms during 24 to 48 h was due to eDNA uptake of C. jejuni cells, resulting in the increased prevalence of dual-resistance mutants.

Campylobacter HGT occurred in intraspecies and dual-species biofilms.

Broiler chickens and retail meat products are usually colonized or contaminated by multiple strains of C. jejuni (41, 42), providing a diverse genetic pool for HGT. To assess the intraspecies genetic exchange, we cultivated the biofilms from two marker strains with different genomic backgrounds (F38011 and NCTC 11168; Table 1) and determined the HGT frequencies using the plating assay. Figure 4 demonstrates the HGT results of four types of biofilms: (i) F38011 Kan^r and Cm^r (F^KF^C), (ii) NCTC 11168 Kan^r and Cm^r (1^K1^C), (iii) F38011 Kan^r and NCTC 11168 Cm^r (F^K1^C), and (iv) NCTC 11168 Kan^r and F38011 Cm^r (1^KF^C). HGT frequencies varied among the different C. jejuni strains. Specifically, C. jejuni NCTC 11168 had significantly higher levels of HGT than F38011 after incubating for 48 h and 72 h (P < 0.05). When F38011 and NCTC 11168 were mixed (i.e., F^K1^C or F^C1^K), their HGT frequencies fell into the range of isogenic strain (i.e., F^KF^C and 1^K1^C) biofilms for up to 48 h. Interestingly, the dual-strain F^K1^C biofilms significantly facilitated the HGT at 72 h postinoculation (P < 0.05) compared to the isogenic strain (i.e., F^KF^C and 1^K1^C) biofilms. At 72 h, the HGT frequencies of F^K1^C biofilms were 381-fold and 9-fold higher than those of F38011 and NTCT 11168 biofilms, respectively. However, the total cell numbers of different biofilm combinations were similar (Fig. S4), indicating that the increased HGT in F^K1^C biofilms was not due to the change of bacterial cell density.

TABLE 1.

Bacteria used for the study of horizontal gene transfer^a

Strain	Source	AMR profile		ARG
		Kanr	Cmr	aphA-3	cat	Reference or source
Campylobacter jejuni
F38011	Human	−	−	–	−	27
F38011 Kanr derivative	Human	+	−	+	−	This study
F38011 Cmr derivative	Human	−	+	–	+	This study
NCTC 11168	Human	−	−	–	−	27
NCTC 11168 Kanr derivative	Human	+	−	+	−	This study
NCTC 11168 Cmr derivative	Human	−	+	–	+	This study
Escherichia coli K-12	Environment	−	−	–	−	27
Salmonella enterica serovar Enteritidis 3512H	Food	−	−	–	−	60

^aAMR, antimicrobial resistance; ARG, antimicrobial resistance gene; +, presence; −, absence. Two AMR profiles were marked in this study, namely, kanamycin resistance (Kan^r) and chloramphenicol resistance (Cm^r).

FIG 4.

Horizontal gene transfer (HGT) in dual-strain C. jejuni biofilms. Different combinations of C. jejuni strains F38011 and NCTC 11168 were cocultivated in 24-well plates at 37°C under microaerobic conditions, including the biofilms containing F38011 Kan^r/Cm^r (FKFC), NCTC 11168 Kan^r/Cm^r (1K1C), F38011 Kan^r/NCTC 11168 Cm^r (FK1C), and NCTC 11168 Kan^r/F38011 Cm^r (1KFC). One-way ANOVA followed by Tukey’s test (equal variance) or Dunnett’s T3 test (unequal variance) was performed to determine the statistical difference among four groups at defined time points (P < 0.05; sample groups with different letters indicate that they have significantly different HGT frequencies). Means and standard deviations (i.e., error bars) were calculated from three independent experiments.

In addition to monospecies biofilms, C. jejuni is believed to survive in the natural environment through colonizing the biofilms that consist of multiple species cultures, such as Escherichia coli (43) and Salmonella enterica (27). To investigate whether dual-species biofilms affect the genetic exchange of C. jejuni, we cultivated C. jejuni F38011 Kan^r and Cm^r strains with the presence of E. coli K-12 or S. Enteritidis 3512H in 24-well plates. HGT frequency and Campylobacter growth profiles are displayed in Fig. 5. C. jejuni F38011 had different trends of HGT when encountering different compositions of biofilm populations. The E. coli-C. jejuni biofilms facilitated the transmission of AMR genes between C. jejuni F38011 Kan^r and Cm^r strains, resulting in an HGT frequency of 2.15 × 10⁻⁸ at 24 h and 3.47 × 10⁻⁸ at 48 h (Fig. 5A). No Kan^r Cm^r dual-resistance transformants were observed in E. coli-C. jejuni biofilms at 72 h (Fig. 5A), which might be due to the induction of viable but nonculturable cells and/or the suppression of C. jejuni growth with the presence of E. coli (Fig. 5B). In comparison, Kan^r Cm^r dual-resistance transformants were identified in the supernatant of E. coli-C. jejuni biofilm system during the incubation period up to 72 h (Fig. 5A). When cocultivated with S. enterica, C. jejuni in biofilm supernatant randomly performed HGT at the early stage of biofilm formation (i.e., 24 h) (Fig. 5C). However, gene transfer in biofilm sessile cells was negligible within 72 h (Fig. 5C), as only limited numbers of C. jejuni cells had culturability (10⁴ to 10⁶ CFU/ml; Fig. 5D). In contrast, there were >10⁷ CFU/ml culturable C. jejuni cells in the monospecies biofilms (Table S2). This result cannot exclude the possibility of HGT in S. enterica-C. jejuni biofilms, considering that the HGT frequency (if there is any) might be below the detection limit of the plating assay (i.e., <1 CFU of transformant per 10⁴ to 10⁶ CFU of total population). Moreover, C. jejuni did not transfer Kan^r or Cm^r genes to another genus, as E. coli and S. enterica cultures obtained from dual-species biofilms could not grow on kanamycin- or chloramphenicol-containing agar plates (data not shown).

FIG 5.

Horizontal gene transfer (HGT) of C. jejuni in dual-species biofilms. Equal concentrations of C. jejuni F38011 (Kan^r and Cm^r) and non-Campylobacter were cultivated in 24-well plates at 37°C under the microaerobic condition. HGT frequency and total Campylobacter concentration were determined for dual-species biofilms containing E. coli K-12/C. jejuni F38011 (A and B) or S. Enteritidis 3512H/C. jejuni F38011 (C and D). For HGT study, Student's t test was conducted to determine the statistical difference between groups (*, P < 0.05). For Campylobacter cell counts, one-way ANOVA followed by Tukey’s test (equal variance) or Dunnett’s T3 test (unequal variance) were performed to determine the statistical difference between groups (P < 0.05). Different lowercase letters indicate significant differences between different ages of biofilm sessile cells, and different uppercase letters indicate significant differences between different ages of biofilm supernatant cells. Means and standard deviations (i.e., error bars) were calculated from four independent experiments.

DISCUSSION

In this study, we constructed a natural transformation model by cocultivating C. jejuni Kan^r and Cm^r strains in biofilms to allow eDNA uptake and chromosomal recombination (Fig. 1). We confirmed the role of transformation in the appearance of Kan^r Cm^r dual-resistance mutants in C. jejuni F38011 biofilms by using PCR (data not shown), spontaneous mutation assay (data not shown), and DNase I treatment assay (Fig. 2). This coculture transformation model has two major advantages for biofilm study over the traditional C. jejuni-DNA model, which was extensively used to investigate natural transformation of C. jejuni in the planktonic state (14, 15, 35, 44). First, the direct addition of gDNA into C. jejuni culture does not fully mimic the entire HGT process in the real environment, as DNA release is the first step of transformation and provides the sources of AMR genetic pools. Second, the rate of natural transformation of gDNA in bacterial biofilms, such as P. aeruginosa, is usually higher than that of eDNA (24). This difference might be due to the degradation of eDNA in bacterial culture, while gDNA remained intact after extraction from commercial kits (24). Using a coculture transformation model could better reflect the temporal dynamics of eDNA generation and degradation in C. jejuni biofilms, leading to a more accurate estimate of HGT frequency.

Almost all previous studies of C. jejuni transformation only focused on investigating planktonic cells. For example, the mixture of C. jejuni 81-176 isogenic Kan^r and Cm^r strains in Bolton broth showed the transformation frequencies ranging from 9.0 × 10⁻¹⁰ to 1.4 × 10⁻⁸ under 10% CO₂ (15). Svensson and others discovered a rapid evolution of transformants after 8 h postinoculation of two C. jejuni 81-176 isogenic AMR strains in MH broth (45). In another study, C. jejuni NCTC 11168 Kan^r and Cm^r strains were cultivated in biphasic MH broth for 5 h, generating dual-resistance transformants at the frequency of 0.028% (16). Our current study demonstrated that the planktonic culture of C. jejuni F38011 Kan^r and Cm^r strains in MH broth did not contribute to detectable transformation after 24 h postinoculation but generated the Kan^r Cm^r dual-resistance mutants after 48 h at the frequency of 10⁻⁸ (Fig. 1). The variation of HGT frequencies in different studies might be caused by the inconsistency in C. jejuni strains and growth media (14).

The current study filled a knowledge gap that C. jejuni had different HGT efficiencies at different modes of growth, including planktonic cells, biofilm sessile cells, and biofilm supernatant cells. Only two studies have reported the transformation of C. jejuni biofilm sessile cells or supernatant culture, and both studies lacked a systematic comparison between biofilms and planktonic lifestyles. Brown and coauthors developed C. jejuni NCTC 11168 and 81116 biofilms for 48 h in the presence of 2 μg of C. jejuni gDNA carrying the Cm^r gene and identified that chloramphenicol-resistant cells were present in both biofilms and biofilm supernatant (29). Bae and colleagues inoculated the mixed culture of C. jejuni NCTC 11168 Kan^r and Cm^r strains into microwell plates and monitored the emergence of Kan^r Cm^r C. jejuni in biofilm supernatant culture (28). Their study demonstrated that the transformation frequency of biofilm supernatant cells reached ∼1.4 × 10⁻⁶ at 48 h, about 6.5-fold higher than that at 24 h (28). Similarly, our study validated that extensive transformation occurred in C. jejuni cocultured biofilms with a frequency up to a magnitude of 10⁻⁶ (Fig. 1). The transformation rate increased as a function of time and stayed stable when biofilms became mature at 48 to 72 h (Fig. 1). Our results support the hypothesis that C. jejuni biofilms performed significantly (P < 0.05) more transformation than planktonic cells regardless of time (Fig. 1). This finding is consistent with other bacterial models, such as P. aeruginosa (24) and Streptococcus spp. (22, 46). Besides, we revealed the mechanism that the presence of transformants in biofilm supernatant culture was due to the continuous shedding of transformants from biofilms (Fig. 1; see also Fig. S2 in the supplemental material). This release model agrees with the findings of a previous study (31). Overall, our study demonstrates that the influence of HGT on C. jejuni AMR evolution is underestimated, as previous studies focused on C. jejuni planktonic lifestyle rather than biofilms.

We discovered that the level of HGT in C. jejuni biofilms was not correlated with cell density, biofilm biomass, and metabolic activity (Fig. 3A to C). This finding is in contrast to the HGT in C. jejuni planktonic cells, which was related to bacterial concentration and growth phase (15, 35). The differences observed in our study of biofilms and the aforementioned studies of planktonic cells might be associated with the use of different transformation models (cocultured cells versus cell-DNA) as well as distinct transcriptional patterns between biofilms and planktonic cells (47). Regarding biofilm matrix, previous studies reported that the gene transfer rate of human pathogen Neisseria gonorrhoeae was independent of biofilm biomass and roughness (48), whereas EPS porosity and biovolume showed intermediate correlations (R = ±0.5, P < 0.05) with the transformation of plasmids in Acinetobacter baylyi biofilms (49). Taken together, other factors remain to be explored to further understand the correlation between C. jejuni HGT and biofilm characteristics in the future, such as biofilm architecture (49) and expression level of transformation-associated genes (50–52).

The eDNA plays a critical role in AMR evolution of C. jejuni. Svensson and coauthors reported that the cultivation conditions that increased DNA release (e.g., with the presence of bile salt) also promoted chromosomal recombination between two C. jejuni strains in shaking liquid cultures (45). On the other hand, DNase I treatment (200 μg/ml) caused a 10-fold reduction in the transfer of chromosome-encoded AMR determinants from Helicobacter pylori to C. jejuni (53). Regarding C. jejuni biofilms, our current study demonstrates that DNase I-treated C. jejuni F38011 biofilms had a significant reduction (P < 0.05) on HGT between F38011 Kan^r and Cm^r strains (Fig. 2), indicating that eDNA is a key vehicle of AMR transmission. The release of eDNA in C. jejuni biofilms could be the result of bacterial autolysis (27, 45). Interestingly, the amount of eDNA in C. jejuni biofilms decreased from 24 h to 48 h (Fig. 3D), whereas the HGT frequencies increased during the same period (Fig. 1). The negative correlation between eDNA and HGT frequencies (Fig. 3E) might be due to multiple eDNAs being expected to be taken up by C. jejuni (14), resulting in fewer eDNAs recovered from biofilms at 48 h. This assumption could also explain the coincidence of unchanged HGT frequency and eDNA amount in C. jejuni biofilms from 48 h to 72 h, when no new HGT event occurring (Fig. 1 and 3D). Moreover, we estimated that approximately one to two C. jejuni genomes were available to one C. jejuni F38011 cells in biofilms (Table S2). This is more than what has been described for C. jejuni planktonic cultures, in which the ratio of extracellular genome to bacterial cells was about 1:100 (35). Compared to the planktonic state, a stronger accumulation of DNA in biofilms is hypothesized to promote more transformation, which was confirmed by our current study (Fig. 1) as well as previous reports (26, 54).

C. jejuni strains have diverse natural competence levels and transformation ability (14). When inoculated with the same C. jejuni chromosomal DNA, different C. jejuni isolates showed strong (10⁻³), relatively weak (10⁻⁴), or even no transformation (14). The distinct transformation performances were partially related to the presence of DNase genes, such as cje0256 (also known as dns), in some C. jejuni genomes (55). In the current study, we tested both C. jejuni strains F38011 and NCTC 11168 and conducted the transformation in biofilms (Fig. 4). Both tested strains lack the three known DNase genes (i.e., dns, cje0556, and cje1441) (56) on their genomes. The HGT frequencies of C. jejuni NCTC 11168 biofilms were significantly higher than that of F38011 biofilms (P < 0.05), with a difference of up to 130-fold (Fig. 4). The different HGT levels were not due to the change of biofilm cell density, as no significant difference (P > 0.05) was identified between different dual-strain combinations (Fig. S4). Future work will be performed to reveal the mechanisms of HGT variation among different strains, such as the transcriptional levels of transformation genes (55). Besides, there was no intraspecies barrier between C. jejuni F38011 and NCTC 11168 for genetic exchange in biofilms (Fig. 4). Cocultivation of F38011 Kan^r and NCTC 11168 Cm^r strains induced significantly higher HGT frequencies (P < 0.05) than biofilms consisting of isogenic strains at 72 h (i.e., a mixture of F38011 Kan^r and Cm^r or a coculture of NCTC 11168 Kan^r and Cm^r) (Fig. 4). This enhanced HGT scenario agreed with the previous observations that C. jejuni could take up chromosomal DNA released from other C. jejuni strains more efficiently than their self-DNA (15), although the mechanisms for this remain unclear. Our data suggest that cocultivation of different C. jejuni strains in the same niches have a synergistic effect on genetic exchange, fostering the transmission of AMR traits.

C. jejuni has been recovered from mixed-culture biofilms in food-related environments, such as broiler chicken carcasses (19, 57), tap water system (20, 58), and chicken houses (18). As a common bacterium in the meat industry, C. jejuni was previously cultured in the mixed-culture biofilms containing E. coli or S. enterica (27, 59, 60). In the current study, C. jejuni F38011 AMR strains developed mixed-culture biofilms with either E. coli or S. enterica. The survival of C. jejuni in dual-species biofilms depended on the type of companion strains. When cocultivating with E. coli, comparable cell counts (∼10⁸ CFU/ml) of C. jejuni and E. coli were identified in biofilms at 24 h, but E. coli started dominating the biofilms after 48 h and the number of C. jejuni cells declined (Fig. 5B, Fig. S5A). In S. enterica-C. jejuni biofilms, the C. jejuni population was about 10- to 1,000-fold less than that of S. enterica during the entire course of incubation (Fig. 5D, Fig. S5B). C. jejuni cells were identified to enter a viable but nonculturable state in the mixed-culture biofilms as incubation time increased (18), possibly explaining the reduction of culturable C. jejuni in the current study.

Although less culturable, C. jejuni was recovered from dual-species biofilms than monococulture biofilms (Fig. 3A and 5). We observed gene transfer between C. jejuni F38011 Kan^r and Cm^r strains in E. coli-C. jejuni biofilms with HGT frequency of ∼10⁻⁸ (Fig. 5A). In comparison, no detectable transformants were obtained in S. enterica-C. jejuni biofilms, whereas random HGT was identified in biofilm supernatant culture at 24 h (Fig. 5C). It is worth noting that the relatively low but significant HGT frequencies in dual-species biofilms still pose concerns to food safety and public health, since C. jejuni transformants may undergo cell division and establish themselves in favorable new niches (61). Furthermore, the lack of detectable HGT in S. enterica-C. jejuni biofilms might be due to the inactivation of C. jejuni, but we cannot exclude the possibility of HGT as the traditional plating assay cannot cultivate the transformants if they enter the viable-but-nonculturable state. In a study conducted by Hausner and Wuertrz, classical plating techniques revealed a 1,000-fold fewer transfer rate than confocal laser scanning microscopy when monitoring the dissemination of green fluorescent protein plasmids from E. coli to soil bacterium Ralstonia eutropha (62). The plating assay is easy to operate and cost-effective, but sacrifices with the relatively low detection limit and the inability of detecting nonculturable cells. To avoid the technical barrier of the plating assay, in situ, real-time studies could be performed in the future by using fluorescence genes as genetic markers and monitoring natural transformation in C. jejuni biofilms through fluorescence imaging coupled with microfluidic “lab-on-a-chip” technique (63).

Antimicrobial selective pressure is not a mandatory driving force for the dissemination of AMR in C. jejuni. Our current study identified prominent transmission of Kan^r and Cm^r genes in C. jejuni monospecies and dual-species biofilms in the absence of antibiotics (Fig. 1, 4, and 5). Qin and coauthors were able to transfer an aminoglycoside resistance genomic island (∼10.6 kb) from C. coli to C. jejuni via natural transformation without the presence of antibiotics, conferring high-level resistance to aminoglycoside antibiotics such as kanamycin and streptomycin (64). The aminoglycoside resistance genomic island remained stable and functional in C. jejuni transformants after 14 passages in antibiotic-free media (64). The spread of neutral and weakly deleterious AMR genes outside antibiotic-selected settings was believed to introduce genetic variation and potentiate adaptation to future antimicrobial challenges in Helicobacter pylori, which belongs to the Campylobacterales order (65). Altogether, the strong ability of C. jejuni to spread and maintain AMR determinants without any selective pressure partially explained the broad and rapid evolution of multidrug resistance among C. jejuni isolates in livestock, sewage, and the human community (4). Once AMR is established, it is highly challenging to completely remove AMR from C. jejuni populations.

In conclusion, our study systematically investigated the HGT in C. jejuni biofilms. Compared to the planktonic state, C. jejuni biofilms demonstrated significantly higher (P < 0.05) HGT frequencies. The factors that impacted HGT frequencies in biofilms included incubation time, C. jejuni strains, and species composition of mixed-culture biofilms. Extracellular DNA played a major role in natural transformation of C. jejuni biofilms, whereas biofilm cell density, biofilm biomass, and cell metabolic activity showed no correlation with the altered HGT frequencies. Moreover, C. jejuni biofilms carried out HGT in the lack of antibiotic selective pressure, indicating that it is able to create a diverse gene pool for genomic recombination, adaptation, and evolution. Our results emphasize that biofilms should be the controlling target for the elimination of C. jejuni AMR in the agrifood continuum and clinical settings. On the other hand, the current study was conducted by incubating the engineering AMR strains under optimal growth conditions. Future studies can further explore the extent of HGT between naturally occurred AMR strains or under different environmental factors (e.g., temperature and oxygen content), mimicking the real-world conditions under which C. jejuni biofilms reside.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Bacterial strains used for the horizontal gene transfer (HGT) study are listed in Table 1. C. jejuni strains were routinely grown either in Mueller-Hinton (MH) broth (BD Difco) with constant shaking at 175 rpm or on MH agar supplemented with 5% defibrinated sheep blood (MHBA) at 37°C under microaerobic condition (85% N₂, 10% CO₂, 5% O₂). Cultivation of Salmonella enterica and Escherichia coli strains was routinely performed at 37°C in Luria-Bertani (LB) broth or on LB agar under aerobic conditions. When necessary, growth media were supplemented with kanamycin (50 μg/ml) and/or chloramphenicol (20 μg/ml).

Construction of C. jejuni antibiotic-resistant mutants.

To monitor the gene transfer between C. jejuni isolates, a kanamycin-resistant (Kan^r) or chloramphenicol-resistant (Cm^r) cassette was inserted into the chromosome of wild-type C. jejuni F38011 and NCTC 11168 through homogenous recombination. The sequence information of Cm^r and Kan^r cassettes referred to previous studies (66–69). The plasmids and bacterial strains involved in the mutant construction are listed in Table S3 in the supplemental material. The primers, as shown in Table S4, were designed using NCBI primer BLAST (National Library of Medicine).

The aphA-3 Kan^r cassette was inserted into the noncoding region between 23S rRNA and 5S rRNA genes on the C. jejuni chromosome. Primers 23SrRNA-F/R were used to amplify a 962-bp fragment containing partial 23S rRNA and its downstream flanking region, while primers 5SrRNA-F/R were used to amplify the 5S rRNA and its upstream flanking region to produce a 947-bp fragment. The aphA-3 gene and its promoter region were amplified using primers aphA3-F/R from the plasmid pRY107, which was extracted from the E. coli pRY107 strain through the Presto Mini plasmid kit (Geneaid, Canada). The target PCR products were separated from nonspecific primer dimers on 1% agarose gel and purified using a QIAquick gel extraction kit (Qiagen, Canada). The vector pUC19 was digested with EcoRI and XbaI, followed by PCR purification through a QIAquick PCR purification kit (Qiagen, Canada). All three PCR fragments and digested pUC19 were ligated and transformed into chemically competent E. coli DH5α cells using a NEBuilder HiFi DNA assembly cloning kit (New England BioLabs, Canada). E. coli harboring the recombinant plasmid pUC19/23S rRNA-Kan^r-5S rRNA was selected on LB agar containing 50 μg/ml kanamycin, followed by plasmid extraction using the Presto Mini plasmid kit (Geneaid, Canada). The presence of 23S rRNA-Kan^r-5S rRNA fragment on the plasmid was confirmed by restriction enzyme digestion using EcoRI and XbaI. The constructed plasmid was introduced into C. jejuni isolates by natural transformation as described previously (70). Transformants were selected on MHBA supplemented with 50 μg/ml kanamycin. The insertion of a Kan^r cassette into C. jejuni chromosome was confirmed by PCR using primers Kan-F/R.

The construction of Cm^r C. jejuni mutants followed a similar procedure. Briefly, a cat Cm^r cassette was inserted into the noncoding region between 16S rRNA and tRNA-Ala genes on the C. jejuni chromosome. PCR was conducted to amplify an 851-bp fragment containing partial 16S rRNA and its downstream flanking region using primers 16SrRNA-F/R, a 967-bp fragment containing tRNA-Ala and its upstream flanking region using primers tRNA-F/R, and a 796-bp fragment containing cat gene and its promoter region using primers cat-F/R. After purification, three PCR products and digested pUC19 were ligated and transformed into E. coli DH5α competent cells using a NEBuilder HiFi DNA assembly cloning kit (New England BioLabs, Canada). Mutants containing the recombinant plasmid pUC19/16S rRNA-Cm^r-tRNA were selected on LB agar containing 20 μg/ml chloramphenicol. The purified plasmid was confirmed by restriction enzyme digestion using EcoRI and XbaI prior to natural transformation into C. jejuni isolates. The presence of the cat gene on the defined chromosomal loci was confirmed by PCR using primers Cm-F/R.

We compared the growth rate, biofilm formation ability, and AMR profiles of parental strains and mutants to assess the potential effect from the insertion of the AMR gene into the bacterial chromosome. Briefly, growth curves of C. jejuni wild type and AMR mutants were monitored by plating assay for up to 72 h. Crystal violet staining assay was used to quantify the biofilm biomass (27, 31). The MICs of each isolate were measured by the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (71).

Biofilm cultivation for HGT.

C. jejuni F38011 Kan^r and Cm^r isolates were used as model strains to study HGT in Campylobacter biofilms. Briefly, an overnight culture of each strain was prepared in MH broth at 37°C under the microaerobic condition for 16 to 18 h. The initial bacterial concentration was adjusted to 2 × 10⁸ CFU/ml in fresh MH broth, followed by mixing Kan^r and Cm^r culture at a volumic ratio of 1:1. One milliliter of bacterial mixture was transferred into each well of polystyrene, tissue culture-treated 24-well plates (Corning, Canada) and incubated statically at 37°C under the microaerobic condition to allow biofilm development. For each sampling time (i.e., 24, 48, 72 h), three wells of biofilm samples were collected and combined to increase the possibility of detecting HGT mutants. To provide sufficient growth nutrients, the supernatant in each well was discarded and replaced by 1 ml of fresh MH broth every 24 h for up to 72 h. Meanwhile, HGT in planktonic cells was used as the comparison standard, where 3 ml of Kan^r/Cm^r mixture was added into a glass culture tube and incubated at constant shaking condition (175 rpm) for up to 72 h.

To further study HGT in intraspecies biofilms, we cultivated different combinations of C. jejuni mixture (1:1, vol/vol) in 24-well plates at 37°C under the microaerobic condition: (i) F38011 Kan^r × NCTC 11168 Cm^r and (ii) NCTC 11168 Kan^r × F38011 Cm^r. Biofilms consisting of either F38011 Kan^r/Cm^r or NCTC 11168 Kan^r/Cm^r were used as the control groups.

To investigate the effect of dual-species biofilms on Campylobacter HGT, we incubated C. jejuni F38011 Kan^r and Cm^r in the presence of E. coli or S. enterica, which commonly coexist with Campylobacter in foods and environmental niches (27, 59, 60). Briefly, overnight cultures of non-Campylobacter strains (i.e., E. coli K-12 or S. enterica serotype Enteritidis 3512H) were incubated in MH broth at 37°C under the aerobic condition for 16 to 18 h, whereas C. jejuni F38011 AMR mutants were inoculated under the microaerobic condition. Bacterial culture of non-Campylobacter and C. jejuni strains was diluted to 2 × 10⁸ and 4 × 10⁸ CFU/ml in fresh MH broth, respectively. We mixed the adjusted culture of non-Campylobacter (either E. coli or S. Enteritidis), C. jejuni F38011 Kan^r, and C. jejuni F38011 Cm^r at a volume ratio of 2:1:1, achieving a dual-species population with each strain at an initial concentration of 1 × 10⁸ CFU/ml. An aliquot of the bacterial mixture (1 ml) was transferred to a 24-well plate and incubated at 37°C under the microaerobic condition for up to 72 h.

Biofilm detachment and quantification.

The emergence of Kan^r Cm^r dual resistance strains was an indicator of HGT. The HGT frequency was calculated by dividing the dual resistance cell counts by total Campylobacter cell counts (28), where bacterial cell counts were quantified by the plating assay. At each sampling time (i.e., 24, 48, and 72 h), we gently withdrew the biofilm supernatant into a collection tube and washed the biofilm using 1× phosphate-buffered saline (PBS; pH 7.4) to remove loosely attached cells. The biofilm was detached from 24-well plates by trypsin as previously described, with minor modifications (72). Specifically, 1 ml of 0.1% trypsin-EDTA solution was added into each well of 24-well plates and incubated at 37°C under the microaerobic condition for 15 min. Both biofilm supernatant and biofilm samples were streaked on MHBA containing 50 μg/ml kanamycin and 20 μg/ml chloramphenicol for the enumeration of Kan^r Cm^r dual resistance cells. When necessary, up to 1 ml of bacterial samples was streaked on the agar plates to achieve a detection limit of 1 CFU/ml. Total Campylobacter cells were counted on MHBA plates. For dual-species biofilm study, total Campylobacter, E. coli, and Salmonella cells were selectively quantified on modified charcoal cefoperazone desoxycholate agar (mCCDA; Sigma, Canada), MacConkey agar (MAC; BD Difco, Canada), and xylose lysine deoxycholate (XLD; BD Difco, Canada), respectively. Campylobacter-related agar plates were incubated at 42°C under the microaerobic condition for 3 days, whereas MAC and XLD plates were grown at 37°C under the aerobic condition for 1 day. No colonies were observed on antibiotic-supplemented MAC or XLD plates (i.e., 50 μg/ml kanamycin and/or 20 μg/ml chloramphenicol), indicating no E. coli or Salmonella Kan^r/Cm^r mutants evolved in this study (data not shown). In other words, all Kan^r/Cm^r colonies grown on MHAB dual-antibiotic agars were supposed to be Campylobacter mutants.

Verification of HGT mutants by PCR.

Colony-directed PCR was applied to detect the presence of aphA-3 and cat genes on the chromosome of Campylobacter Kan^r/Cm^r mutants. For each sample, a total of 3 single bacterial colonies were randomly collected from MHBA dual-antibiotic plates. Every single colony was suspended in 3 μl of DNase-free sterile water and used as the source of template DNA for PCR. Primers Kan-F/R and Cm-F/R (Table S4) were used for the detection of aphA-3 and cat, respectively. The PCR was performed in a 25-μl volume containing 1 μl of bacterial suspension, 10 μM forward and reverse primers, and 1× Taq FroggaMix (FroggaBio, Canada). The amplification was initially incubated at 95°C for 3 min, followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, and 2.5 min at 68°C, and then we carried out a final extension at 68°C for 10 min. DNase-free sterile water was used as the negative control and gDNA from C. jejuni Kan^r and Cm^r mutants as the positive controls. PCR products were confirmed by 1% agarose gel electrophoresis.

Assessment of spontaneous mutation in biofilms.

Campylobacter biofilm has been reported to show enhanced mutability associated with AMR (e.g., fluoroquinolone resistance) under optimal growth conditions and without antibiotic selection stress (73). In this study, we also assessed the spontaneous mutation associated with Kan^r or Cm^r in biofilms. Briefly, overnight cultures of C. jejuni Kan^r or Cm^r were suspended in fresh MH broth to give an initial concentration of 1 × 10⁸ CFU/ml. Sterile 24-well plates were inoculated with 1 ml of single-strain culture and incubated at 37°C under the microaerobic condition for up to 72 h. Fresh MH broth was supplied every 24 h to replace the growth medium in 24-well plates. Biofilm samples were collected at 24, 48, and 72 h and streaked onto MHBA dual-antibiotic plates (50 μg/ml kanamycin and 20 μg/ml chloramphenicol) to monitor the emergence of Kan^r Cm^r dual resistance strains.

DNase I treatment.

To identify the role of eDNA on biofilm HGT, we treated C. jejuni biofilms with DNase I, as this endonuclease can nonspecifically cleave DNA and reduce its availability. Briefly, overnight cultures of C. jejuni F38011 Kan^r and Cm^r strains were separately diluted to 2 × 10⁸ CFU/ml in fresh MH broth and thoroughly mixed at a volume ratio of 1:1. The bacterial cocktail was supplemented with 100 U/ml DNase I (Thermo Scientific, Canada). One milliliter of bacterial culture was deposited into a 24-well plate and incubated statically at 37°C under the microaerobic condition for 48 h. Growth media were routinely replaced every 24 h with fresh MH broth containing 100 U/ml DNase I. In parallel, C. jejuni biofilm without the addition of DNase I was regarded as the negative control. HGT frequencies of DNase-treated and nontreated biofilms were determined by the plating assay.

Crystal violet staining assay.

Biofilm biomass was quantified by crystal violet staining assay according to the previous studies, with modifications (31). Briefly, biofilms grown on 24-well plates were gently rinsed twice with water and heat-fixed at 60°C for 30 min, and then 1 ml of 1% crystal violet solution (Ricca, Canada) was added into 24-well plates and incubated at room temperature for 30 min. The stained biofilms were gently washed with water to remove unbound crystal violet dye. After drying at 37°C for 10 min, bound crystal violet was dissolved in 1 ml of 95% ethanol solution. Samples (100 μl) were transferred to a clean 96-well plate (Corning, Canada), and the absorbance at 595 nm was measured by a Tecan Spark multimode microplate reader (Tecan Life Sciences).

TTC assay.

TTC assay was performed to measure the metabolic activity of biofilm sessile cells as previously described, with modifications (36). After cultivating biofilms in 24-well plates for 24 to 72 h, we discarded the supernatant and rinsed the biofilms with 1× PBS. One milliliter of 0.01% TTC in MH broth was added to biofilm-containing wells, followed by static incubation at 37°C under the microaerobic condition for another 24 h. The TTC solution was discarded, and biofilms were treated with 1 ml of ethanol-acetone mixture (8:2, vol/vol) at room temperature for 5 min to dissolve the TTC reaction products. An aliquot of 200 μl sample was transferred to a 96-well plate, and the absorbance was measured by a Tecan Spark multimode microplate reader at a wavelength of 500 nm.

Quantification of biofilm eDNA.

The amount of eDNA in biofilm was quantified using SYBR green I dye (Invitrogen, Canada) according to a previous protocol, with modifications (27). After incubating biofilms for certain time periods (i.e., 24, 48, and 72 h), we discarded the growth media and detached biofilms from 24-well plates using 300 μl of 0.1% trypsin-EDTA solutions. Biofilms were collected into clean microcentrifuge tubes, followed by centrifugation at 15,000 × g for 3 min. An aliquot of supernatant (95 μl) was transferred into a well of black 96-well plates (Greiner Bio-One, Canada) and mixed with 5 μl of SYBR green I working solution, as prepared by diluting the stock solution 100 times in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0; Invitrogen, Canada). The plate was incubated in a Tecan Spark multimode microplate reader with orbital shaking for 5 min. The fluorescence signal was recorded in the microplate reader with an excitation light centered at 485 nm (±10 nm) and the emission centered at 535 nm (±10 nm). To calculate the eDNA amount, a standard curve was generated by detecting the fluorescence signal of lambda DNA (Invitrogen, Canada), ranging from 0.125 μg/ml to 2.5 μg/ml in 0.1% trypsin-EDTA solutions.

Statistical analyses.

All experiments were independently performed at least three times. Results are shown as the means ± standard deviations. Statistical analysis was conducted using SPSS Statistics 22.0 (IBM). The criteria of selecting appropriate statistical analyses included group number, sample size, and the variance of data (74). Student's t test was applied to determine the statistical difference (P < 0.05) between two groups, while one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test (equal variance), Games-Howell test (unequal variance; when sampling sizes were 6), or Dunnet’s T3 test (unequal variance; when sampling sizes were <6) was used to determine the statistical differences among multiple groups (n ≥ 3). Pearson correlation analysis was performed to assess the correlation between HGT frequency and biofilm eDNA availability. GraphPad Prism 7 (GraphPad Software) was used for graphing.