Development, Stability, and Molecular Mechanisms of Macrolide Resistance in Campylobacter jejuni

Bacterial strains and culture conditions. C. jejuni NCTC 11168, an Ery^s strain (MIC = 1 μg/ml), was purchased from ATCC (catalogue no. 700819) and was used as a parent strain in this study selecting for macrolide-resistant mutants in vitro. NCTC 11168 was also used in a recent chicken study to select Ery^r mutants in vivo (16), from which a panel of individual Ery^r mutants (strains DC1 to DC20) isolated from chickens (Table 1; see Table 3) were used in this study. A new in vivo-selected Ery^r mutant, named DC26 (Ery MIC = 64 μg/ml), was derived from DC2 and isolated from one bird on day 43 in this study (experiment A) (Table 1). DC26 was further used for selecting Ery^r mutants with higher Ery resistance in this study (experiment B) (Table 1). The Campylobacter strains were grown routinely on Mueller-Hinton agar (MHA) plates or in Mueller-Hinton broth (MHB) at 42°C under microaerobic conditions. Campylobacter-specific growth supplements and selective agents (SR084E and SR117E; Oxoid) were added to the media when needed. When required, the MH media were also supplemented with various amounts of Ery.

Long-term exposure of low-level Ery^rCampylobacter mutants to a subtherapeutic dose of tylosin in chickens.Two independent experiments (experiments A and B) were conducted, and the experimental design is detailed in Table 1. For both trials, day-old broiler chicks (a gift from the commercial company Hubbard Hatchery, Pikeville, TN) were randomly assigned to two treatment groups. All birds were placed in sanitized wire cages with unlimited access to feed and water. All feed was prepared by the feed mill at the Johnson Animal Research and Teaching Unit. Medicated feed containing tylosin phosphate (Elanco Animal Health) was prepared, in accordance with the label for preparation of medicated feed used for growth promotion of chickens, to obtain a final tylosin concentration of 50 mg/kg. Prior to inoculation with C. jejuni, all birds were confirmed to be free of Campylobacter by culture of cloacal swabs. At 15 days old, all birds in each group were inoculated with fresh C. jejuni DC2 or DC26 culture (10⁷ CFU/bird) via oral gavage. Because a distinct characteristic of C. jejuni colonization in poultry is that this organism is not detected in chickens less than 2 to 3 weeks old under commercial broiler production conditions, inoculation of birds with C. jejuni at 15 days old in these two trials should allow us to measure the impact of using tylosin as a growth promoter in the development of Ery resistance in Campylobacter under conditions similar to those of commercial production. After the birds were inoculated, cloacal swabs were collected from each chicken at different time points until the end of each experiment (Table 1). Samples from each bird were diluted serially and spread onto three different types of MHA plates to recover total Campylobacter populations (normal selective plates), Ery^r populations (selective plates containing 8 μg/ml of Ery), and highly Ery^r populations (selective plates containing 128 μg/ml of Ery). Campylobacter colonies were counted following 48 h of incubation at 42°C under microaerobic conditions. Individual colonies were collected from selective plates for each chicken and were used for MIC testing as described below. Multiple isolates with different Ery MICs were analyzed by PCR to confirm their genetic identities. The PCR analysis was done using primers specific for the cmp gene encoding the major outer membrane protein, as described previously by Huang et al. (13), and revealed no difference between input strain and output isolates.

In vivo stability of Ery resistance using the chicken model system.The in vivo stability of Ery resistance was evaluated using the chicken model system, and the experimental design is detailed in Table 1 (experiment C). The bird source and maintenance are the same as those for experiments A and B as described above. However, chickens in both groups received nonmedicated feed throughout the trial. To maximize exposure time to antibiotic-free feed, all birds were inoculated with approximately 10⁷ CFU fresh C. jejuni culture via oral gavage at 3 days old. Prior to inoculation with C. jejuni, all birds were confirmed to be free of Campylobacter by cultured cloacal swabs. The mutants DC2 and DC6, with different levels of Ery resistance, were used for evaluation of in vivo Ery resistance stability in this study (Table 1). After the birds were inoculated, cloacal swabs were collected at different time points. Isolation of Campylobacter spp. and differential plating for enumerating the proportion of the mutant colonies were conducted as described above. Representative isolates from each chicken were selected for the Ery MIC test.

Detection limit and statistical analysis.In all chicken experiments, the detection limit of the plating methods was approximately 100 CFU/g of feces. Student's t test was used to examine the significant difference in Campylobacter colonization levels (log transformed CFU/g feces) at each sampling point between the two treatment groups. A P value of <0.01 was considered significant.

In vitro stability of Ery resistance.The same Ery^r mutants DC2 and DC6 were also used for the in vitro stability test. Briefly, the two mutants were inoculated in antibiotic-free MHB and grown in microaerophilic conditions at 42°C. The Campylobacter culture was subcultured every 2 to 3 days in 4 ml fresh MHB (1:400 dilution) for 81 days in the absence of any antibiotics. Following passages 10, 20, and 33, the cultures were serially diluted (10-fold dilutions) in MHB and plated on both MHA plates and MHA plates supplemented with Ery at a final concentration of 8 μg/ml (for DC2 and DC6) or 128 μg/ml (for DC6). Plates were incubated at 42°C under microaerophilic conditions for 48 h. Total numbers of colonies on each type of plate were counted and compared. In addition, following passage 33 differential plating, 10 colonies for each mutant were randomly chosen from MHA plates and were subjected to the Ery MIC test as described below.

Antibiotic susceptibility test.MICs of antibiotics were tested using the agar dilution method as recommended by the CLSI (7). For quality control, C. jejuni ATCC 33560 and a quality control range for an Ery MIC of 0.25 to 2 μg/ml were used in this study. According to the breakpoints recommended by the CLSI (7), Ery MICs of ≤8 μg/ml and ≥32 μg/ml are considered susceptible and resistant, respectively. In this report, unless specifically defined, high-level Ery resistance or highly Ery^r mutants corresponded to an Ery MIC of >512 μg/ml in C. jejuni. Ery was purchased from Sigma Chemical Co., St. Louis, MO.

Selection of macrolide-resistant mutants in vitro.Erythromycin or tylosin tartrate (MP Biomedicals) was used as the selective agent for selecting spontaneous macrolide-resistant mutants in vitro. Briefly, Ery^s NCTC 11168 culture was grown in antibiotic-free MHA plates to the late logarithmic phase. The cultures were plated on increasing concentrations of Ery and tylosin (fourfold to 16-fold the initial MIC of NCTC 11168). Following 3 to 5 days of incubation under microaerophilic conditions at 42°C, a single macrolide-resistant colony on selective plates was selected, cultured in MHB, and stored at −80°C. The Ery^r and tylosin-resistant mutants obtained from the first round of selection were grown in MHB containing sublethal concentrations of Ery (i.e., 4 μg/ml for JL276 and 8 μg/ml for JL290) or tylosin (2 μg/ml for JL295) (see Table 4), respectively, and then plated on selective plates again to select mutants with a higher level of resistance. If needed, the procedure was repeated up to four times to obtain highly macrolide-resistant mutants. All in vitro-selected mutants were subjected to the MIC test using the standard agar dilution method as described above. In addition, genomic DNA prepared from each mutant was used for both PCR amplification and sequence analysis of the 23S rRNA gene and the ribosomal protein L4 and L22 genes as described below.

Sequence analysis of the 23S rRNA gene and ribosomal protein L4 and L22 genes.Domain V of the 23S rRNA gene sequence of C. jejuni was amplified by PCR with gene-specific primers (5′-GTAAACGGCGGCCGTAACTA-3′ and 5′-GACCGAACTGTCTCACGACG-3′) (14). Ribosomal protein L4 gene-specific primers 5′-GTAGTTAAAGGTGCAGTACCA-3′ and 5′-GCGAAGTTTGAATAACTACG-3′ and L22 gene-specific primers 5′-GAATTTGCTCCAACACGC-3′ and 5′-ACCATCTTGATTCCCAGTTTC-3′ were used to amplify the complete L4 gene rplD and the L22 gene rplV, respectively (6). The PCR conditions were as follows: 94°C for 5 min, 94°C for 30 s, 52°C for 30 s, and 72°C for 45 s for 35 cycles and a final extension at 72°C for 10 min. The amplified PCR products were purified with the QIAquick PCR purification kit (Qiagen) prior to being sequenced. DNA sequences were determined in the DNA facilities at the University of Tennessee. Sequence analysis was performed using the DNAStar package.

Insertional mutagenesis of the cmeB gene in various isolates.Natural transformation was used to construct the isogenic cmeB mutants of various Campylobacter isolates as described previously (16).

Colonization of Ery^r mutants in response to tylosin treatment.In both experiments A and B, all chickens were successfully colonized with C. jejuni as early as day 17 (for experiment A) or day 20 (for experiment B). In experiment A, the shedding level of Campylobacter jejuni in feces was consistently higher (up to 1.8 log₁₀ units) in the chickens given nonmedicated feed than in those given tylosin-containing feed, except that on day 43 and day 50 the shedding levels of C. jejuni for the nonmedicated group were slightly lower (P > 0.05) than those from the medicated group (Fig. 1A). In experiment B, chickens were assigned to two groups that were inoculated with DC2 or DC26 and all chickens were treated with medicated feed throughout the study. As shown in Fig. 1B, both groups of chickens displayed similar shedding levels of Campylobacter jejuni throughout the study, although DC2 seemed colonize slightly better than DC26 on day 20.

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FIG. 1.

Levels of Campylobacter colonization in chickens. (A) Shedding levels of C. jejuni DC2 in chickens receiving nonmedicated feed and feed supplemented with the growth promoter tylosin (50 mg tylosin/kg feed), corresponding to experiment A (Table 1). (B) Shedding levels of C. jejuni DC2 or DC26 in chickens receiving feed supplemented with the growth promoter tylosin (50 mg tylosin/kg feed), corresponding to experiment B (Table 1). (C) Shedding levels of low-level Ery^r DC2 or high-level Ery^r DC6 in chickens receiving nonmedicated feed, corresponding to experiment C (Table 1). Each bar represents the mean log₁₀ CFU/g feces ± the standard deviation in each group. Different letters above the bars on each sampling day denote a significant difference (P < 0.01).

Effect of prolonged low-dose tylosin treatment on the emergence of high-level Ery^rC. jejuni in chickens.In experiment A, in the absence of tylosin selection pressure (group 1, the control), the Ery MICs of isolates gradually decreased and returned to the wild-type level of susceptibility (Table 2), suggesting that low-level Ery resistance cannot persist for a long time in vivo in the absence of tylosin selection pressure. Specifically, when the chickens were 29 days old (14 days after inoculation), all isolates were susceptible to Ery, with Ery MICs of 4 μg/ml, which was also confirmed by the differential plating method (data not shown). Supplementation of tylosin in feed maintained the Ery resistance phenotype of DC2 throughout the study (Table 2). In addition, Ery^r mutants with increased MICs (64 μg/ml) emerged at different sampling days after continuous exposure to low doses of tylosin (Table 2). The twofold MIC increase in these Ery^r mutants is not caused by methodological error and has been confirmed by an independent MIC test. However, as long as 35 days of extended exposure of DC2 to low doses of tylosin did not select highly Ery^r mutants in experiment A (Table 2).

To further confirm the findings from experiment A, DC2 and its new in vivo derivative DC26 were used to challenge chickens receiving tylosin-containing feed. As shown in Table 2, regardless of the mutants used for inoculation, none of the chickens shed high-level Ery^rC. jejuni (MIC > 256 μg/ml) during 33 days of continuous exposure to low doses of tylosin, which was also confirmed by differential plating (data not shown). However, Ery^r mutants with increased resistance emerged in both groups of chickens. For example, a mutant with an Ery MIC of 256 μg/ml was obtained on day 48 in group 1 (Table 2). In group 2, which was inoculated with the DC26 mutant, the mutants with an Ery MIC of 128 μg/ml were selected on each sampling day (Table 2). Representative isolates obtained from experiments A and B are given in Table 3 and were used for examination of molecular mechanisms of Ery^r in C. jejuni as described below.

In vivo stability of Ery resistance in C. jejuni.All chickens were successfully colonized by C. jejuni DC2 or DC6 by as early as day 12 (9 days after inoculation) (Fig. 1C). An MIC test of all randomly selected isolates from each individual chicken at each time point showed that in vivo stability of Ery resistance was affected by the resistance level (Fig. 2A). For low-level Ery^r mutant DC2, by day 22, 25% of the isolates displayed an Ery MIC of 4 μg/ml and the majority of isolates showed reduced Ery MICs (16 μg/ml). In fact, the instability of low-level Ery resistance was also observed in chickens as early as 9 days after inoculation (day 12); 2 of 12 isolates from 12-day-old chickens already displayed reduced Ery MICs (16 μg/ml). With further growth of DC2 in chickens without tylosin selection pressure, all isolates recovered on day 38 and day 47 were susceptible to Ery, with MICs comparable to those of the wild-type Ery^s strain (Fig. 2A). In contrast, all isolates from chickens inoculated with DC6 consistently displayed high levels of Ery resistance, regardless of the sampling date (Fig. 2A). These MIC results were also consistent with differential plating results, which showed that approximately 100% of C. jejuni populations from chickens inoculated with DC6 grew on selective MHA plates containing 128 μg/ml of Ery for the entire study but that no Ery^rC. jejuni mutant was detected in DC2-inoculated chickens by day 38 using selective MHA plates containing 8 μg/ml Ery (data not shown).

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FIG. 2.

Stability of Ery resistance of low-level Ery^rC. jejuni DC2 (MIC = 32 μg/ml) and high-level Ery^rC. jejuni DC6 (MIC > 512 μg/ml). (A) Stability of Ery resistance in vivo. Chickens in each group were inoculated with DC2 or DC6 via oral gavage at 3 days old. Chickens received nonmedicated feed throughout the study. The percentage of the Ery^r population for DC2 was calculated based on the MIC using a breakpoint of >8 μg/ml, and the percentage of the Ery^r population for DC6 was calculated based on the MIC using a breakpoint of >512 μg/ml. (B) Stability of Ery resistance in vitro. The percentage of the Ery^r population for DC2 was calculated based on differential plating using plates containing 8 μg/ml of Ery, and the percentage of the Ery^r population for DC6 was calculated based on differential plating using plates containing 128 μg/ml of Ery.

In vitro stability of Ery resistance in C. jejuni.As shown in Fig. 2B, more than 60% of DC2 populations could still be selected on MHA plates containing 8 μg/ml of Ery after 10 passages in the absence of antibiotic selection pressure, while all DC6 populations grew well on MHA plates containing 128 μg/ml of Ery. By passages 20 and 33, only 13% and 7% of DC2 populations were selected on MHA plates containing 8 μg/ml Ery, respectively (Fig. 2B). However, DC6 still displayed high stability after passages 20 and 33; approximately 100% of the population could still be recovered on MHA plates containing 128 μg/ml of Ery (Fig. 2B). These differential plating results were also confirmed by Ery MIC tests of representative isolates selected on MHA plates after passage 33 (10 isolates per strain). The majority of DC2 isolates (6 of 10) showed an Ery MIC of 2 μg/ml, which is comparable with the MIC of the Ery^s parent strain, and four other isolates displayed an Ery MIC of 8 μg/ml. However, 10 randomly selected DC6 isolates all displayed an Ery MIC of >512 μg/ml, indicating that high levels of Ery resistance are extremely stable in C. jejuni in vitro.

In vitro selection of macrolide-resistant mutants.The representative macrolide-resistant mutants selected in vitro are listed in Table 4. Four low-level Ery^r mutants (JL276 to JL279) were obtained from the initial selection by using Ery as the selective agent. The second round of selection using JL276 resulted in Ery^r mutants with increased MICs, such as JL283 to JL286. The highly Ery^r mutants (JL287 to JL289) were successfully obtained during the third round of selection using the JL286 strain. These highly Ery^r mutants displayed colony sizes on MHA containing 128 μg/ml of Ery similar to those on the MHA plates. In an independent selection experiment, although we also obtained low-level Ery^r mutants (JL290 to JL294) in the first round of selection and intermediate-level Ery^r mutants (JL299 and JL300) in the second round using JL290, we failed to obtain highly Ery^r mutants using JL299 as the parent strain even if the stepwise selection was repeated two more times. Highly Ery^r mutants also failed to be obtained from JL290 in other independent experiments, from which strains JL305 to JL308 were selected for analysis (Table 4). When tylosin was used as the selective agent, low-level Ery^r mutants (JL295 to JL298) were obtained in the initial selection step. The Ery^r mutants with much higher MICs (JL301 to JL304) were obtained from the second round of selection using JL295 grown in the presence of a sublethal concentration of tylosin. However, we failed to obtain highly Ery^r mutants using JL302 for selection in two independent experiments.

Molecular mechanisms of Ery resistance. (i) Analysis of 23S rRNA gene sequences.Thirty-nine in vivo-selected and 30 in vitro-selected Ery^rC. jejuni mutants (all NCTC 11168 derivatives) were subjected to PCR amplification of the 23S rRNA gene and rplD and rplV genes encoding ribosomal proteins L4 and L22, respectively. The sequences of these PCR products were aligned to the corresponding sequence of parent strain NCTC 11168 to identify the specific mutations that occurred in the ribosome.

With respect to the 23S rRNA gene, all highly Ery^r mutants isolated from chickens (DC3, -6, -9, -31, -32, and -33) displayed the same A2074G point mutation in the 23S rRNA gene but none of the rest of the 33 isolates with Ery MICs up to 256 μg/ml showed point mutations in the 23S rRNA gene (Table 3), which is consistent with our previous examinations of some in vivo-selected isolates (16). Isolates DC31, -32, and -33 are probably identical to DC6, the strain used for inoculation in experiment C in this study (Table 1). DC3, -6, and -9 were isolated from different birds in a previous study (16). Interestingly, all three in vitro-selected high-level Ery^r mutants (JL287 to -289) displayed different point mutations in the 23S rRNA gene, with an A→C point mutation at position 2074 (Table 4). According to sequence results, it appeared that the A2074C mutation was present in two of the three copies of the 23S rRNA gene in mutants selected in vitro, because the sequence chromatogram showed double peaks in the same position, where the C peak was two times higher than the A peak. Notably, Ery was used as a selective agent to select these three in vitro Ery^r mutants. To determine if different selective agents lead to different 23S rRNA gene mutations, we also used tylosin as a selective agent to select Ery^r mutants in vitro. However, we failed to obtain highly Ery^r mutants by using tylosin as the selective agent, and none of the tylosin-selected mutants displayed point mutations in the 23S rRNA gene, even if the Ery MIC was as high as 512 μg/ml (Table 4).

(ii) Analysis of ribosomal protein L4 gene sequences.Changes in ribosomal protein L4 (encoded by the rplD gene) were not detected in mutants selected in vitro (Table 4) but were observed in the majority of in vivo-selected Ery^r mutants with an Ery MIC of ≤256 μg/ml (Table 3). It is important to mention that all isolates obtained from experiment A, DC39 from experiment B, and DC28 to DC30 from experiment C (Table 3) are directly related to DC2, the strain used for inoculation. Three specific changes in L4 were observed in these in vivo-selected isolates. Many mutants harbored a G74D mutation in L4 that was also recently reported by Cagliero et al. (6). A G-to-A transition was found at nucleotide 170 of the rplD gene sequence in four in vivo-selected isolates (DC10, DC14, DC17, and DC19), which led to a Gly-to-Asp modification at position 57 of the L4 protein (Table 3). Another novel mutation is a G-to-T transition at nucleotide 170 of the rplD gene, which led to the Gly-to-Val modification at position 57 of the L4 protein in mutant DC39 (Table 3). DC39 showed a higher Ery MIC (256 μg/ml) than that of its parent strain, DC2. Notably, DC39 still carried a G74D mutation in L4 from DC2, which may work together with the new G57V mutation, conferring a higher level of Ery resistance.

(iii) Analysis of ribosomal protein L22 gene sequences.Modification in ribosomal protein L22 (encoded by the rplV gene) was observed only in the Ery^r mutants selected in vitro but not in the mutants selected in vivo (Tables 3 and 4). Specifically, a nine-base (ACTTCACAT) tandem duplication at position 292 in the rplV gene, which led to the insertion of three amino acids (TSH) at position 98 (Ins₉₈TSH) in protein L22, was observed in in vitro-selected isolates whose Ery MICs ranged from 32 to 512 μg/ml (Table 4). The choice of a specific selective agent (Ery or tylosin) did not affect the development of Ins₉₈TSH modification in the L22 protein (Table 4).

(iv) Inactivation of CmeABC multidrug efflux pump.To determine the contribution of the CmeABC efflux pump to the acquired Ery resistance, a CmeABC mutation was transferred to selected Ery^r mutants, and the Ery MIC of each isogenic CmeB mutant was measured by the standard agar dilution method. Regardless of the presence of a specific mutation in the 23S rRNA gene (e.g., DC32, JL287, and JL288), in the L4 protein (e.g., DC22 and DC27), or in the L22 protein (e.g., JL290, JL301, and JL303) or the absence of any mutation (JL283 and JL284), inactivation of cmeB dramatically reduced the Ery MIC (8- to 1,024-fold) compared to that of its parent strain (data not shown). This observation confirmed previous findings that CmeABC works synergistically with other mechanisms to maintain high-level and low-level Ery resistance in C. jejuni (6, 16).