Nationwide Stepwise Emergence and Evolution of Multidrug-Resistant Campylobacter jejuni Sequence Type 5136, United Kingdom

We examined whole-genome–sequenced Campylobacter jejuni and C. coli from 2012–2015 isolated from birds and human stool samples in North East Scotland for the presence of antimicrobial resistance genes. We found that sequence type (ST) 5136 (clonal complex 464) was the most prevalent multidrug-resistant strain of C. jejuni exclusively associated with poultry host reservoirs and recovered from human cases of campylobacteriosis. Tetracycline resistance in ST5136 isolates was due to a tet(O/32/O) mosaic gene, ampicillin resistance was conferred by G → T transversion in the −10 promoter region of blaOXA-193, fluoroquinolone resistance was due to C257T change in gyrA, and aminoglycoside resistance was conferred by aac. Whole-genome analysis showed that the strain ST5136 evolved from ST464. The nationwide emergence of ST5136 was probably due to stepwise acquisition of antimicrobial resistance genes selected by high use of β-lactam, tetracycline, fluoroquinolone, and aminoglycoside classes of drugs in the poultry industry.

infections are mild and self-limiting and resolve within a few days, but severe or prolonged infections can occur, particularly in the young, elderly, and immunocompromised patients with AIDS or other vulnerable categories for which therapeutic intervention may be warranted (8).
For clinical therapy of campylobacteriosis, antimicrobial drugs, such as erythromycin, are usually prescribed, but ciprofloxacin is advised in moderately severe cases of nonconfirmed gastroenteritis and for travelers' diarrhea (9). In recent years, treatment with fluoroquinolones has been challenging because of an increasing prevalence of fluoroquinolone resistance in human Campylobacter isolates that led to the ban of the fluoroquinolone enrofloxacin for use in poultry in the United States in 2004 (10).
The Veterinary Medicines Directorate reports that in 2016 a total of 337 tons (17% decrease from 2015) of authorized veterinary antimicrobial drugs were sold in the United Kingdom. The sales of trimethoprim, sulphonamides, β-lactams, and aminoglycosides remained stable between 2012 and 2016, but notable reductions were observed for tetracycline (30%), macrolides (24%), and fluoroquinolones (29%) from 2015 to 2016 (11). Only enrofloxacin and difloxacin are licensed for poultry use; 0.5 metric tons of active ingredient were sold for use in poultry production in 2015 to the British Poultry Council, a national trade group that accounts for 90% of all broilers produced in the United Kingdom (12).
The main drivers for the acquisition of antimicrobial resistance (AMR) are selection pressure and the opportunity for horizontal gene transfer (13). Multidrug resistance has been defined as resistance to >3 classes of antimicrobial drugs and can occur by stepwise mutation or single plasmid acquisition of AMR genes (14,15). A report commissioned by the UK Food Standards Agency shows that drug-resistant Campylobacter species are becoming more prevalent; evidence shows increasing levels of resistance in bacteria from poultry meat (16), which makes up around half of all meat by weight purchased in the United Kingdom (33 kg/person) (17).
The aim of this study was to establish the evolutionary course of events that led to the emergence of a multidrugresistant (MDR) organism by considering the interplay between selection pressures and genetic changes. Specifically, we investigated whether antimicrobial resistance to β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides in the ancestral lineages of C. jejuni ST5136 (Figure 1) led to the emergence of a strain that is the causative agent of human campylobacteriosis in 1 of 20 cases in the United Kingdom (18).

Bacterial Isolates and Genotyping
Campylobacter isolates for this study came from patients who had campylobacteriosis during 2012-2015 and from chicken and turkey retail meat samples. We incubated the meat samples at ambient temperature in enrichment broth for 1 h with occasional agitation and plated 100 μL of the broth on mCCDA plates (E & O Laboratories, http:// www.eolabs.com). We then incubated the plates under microaerobic conditions at 37ºC for 48 h and extracted genomic DNA using the Wizard Genomic DNA Purification Kit (Promega, https://www.promega.com). Wholegenome sequencing was performed using HiSeq 2000 sequencer (Illumina, https://www.illumina.com) with 100 bped-end sequencing. We assembled FASTQ paired-end reads using Velvet (19) with a typical 50× coverage and assembled genome size of ≈1.6 Mbp. We uploaded the genomes to the Bacterial Isolate Genome Sequence Database (BIGSdb) and typed them using the 7-locus multilocus sequence typing (MLST) and whole-genome MLST (wgMLST) schemes (20,21).

Genome Annotation and Phylogenetic Analyses
We chose 100 isolates with 1 C. coli (CC828) outgroup that were representative of the closely related clonal complexes CC464, CC353, CC354, and CC574 from BIGSdb. The group consisted of isolates from North East Scotland (n = 31), England (n = 68), and Estonia (n = 1). We annotated their genomes using RAST (26) and analyzed them by whole-genome multilocus sequence typing (wgMLST) using 1,643 Gundogdu loci (27). We constructed a phylogenetic tree in Bionumerics v. 7.6 (Applied Maths, http://www.applied-maths.com) using the topscore unweighted pair group method with arithmetic mean algorithm based on similarity of wgMLST loci. We constructed maximum-likelihood trees for aminoglycoside resistance determinants in MEGA7 (28).

Antimicrobial Susceptibility Testing and Strain Stability
We determined antimicrobial susceptibility for 21 representative CC464 isolates with genotypic polymorphisms across the range of detected resistance markers. We used Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/L β-nicotinamide adenine dinucleotide for antimicrobial susceptibility testing using the disk diffusion method for ciprofloxacin (5 μg/disk), gentamicin (10 μg/disk), tetracycline (5 μg/disk), kanamycin (5 μg/disk), amikacin (30 μg/disk), tobramycin (10 μg/diskc), and streptomycin (30 μg/disk) (29,30). We determined MIC of ampicillin (AMP) by Etest (Oxoid, http://www.oxoid.com) and interpreted the MIC AMP and disk susceptibility results using guidelines from the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute and interpreted them as described previously (29). We used the antimicrobial drug-susceptible isolate ARI3025 as a control. We subcultured the ST5136 isolate ARI4158 on antibioticfree blood agar plate (ARI4158_20) 22 times to assess the stability of its antimicrobial resistance genes.

Statistical Analyses
We calculated 95% CIs by 10,000 bootstrap iterations to assess if there were significant differences in clonal complexes or individual strains with respect to resistance to different antimicrobial drug categories or AMR resistance alleles. If the CIs did not overlap (e.g., between 2 groups for a particular factor), then we inferred the comparison to be statistically significant (p<0.05).

Antimicrobial Susceptibility Testing and Strain Stability
We performed susceptibility testing and corroborated 21 isolates of CC464 with genotypes predictive of resistant or sensitive phenotypes for different antimicrobial drugs (Table). Past studies show that disk diffusion correlated well to MIC determination and reliably distinguished between sensitive, intermediate, and resistant isolates of Campylobacter (33); hence, we used this method to assess and interpret genotypic data linked to isolate phenotype. Previous research has observed that the C257T gyrA genotype conferred ciprofloxacin resistance, G57T change in the -10 promoter of bla OXA-61 -like led to an increase in ampicillin resistance, and all tetracycline resistance genes conferred resistance to tetracycline (Table) (22,34,35). We observed resistance to kanamycin in 18/21 aac3-positive isolates harboring alleles v22 and v17, indicating that these variants may have the ability to hydrolyze kanamycin (Table); this finding warrants further investigation. Most of the isolates displayed a tobramycin-sensitive phenotype, whereas all were sensitive to amikacin, streptomycin, and gentamicin (Table). We subcultured isolate ARI4158 (ST5136) 22 times on antibiotic-free blood agar to assess the stability of its antimicrobial resistance genes, but we found it to be resistant like the parent isolate.

Phylogenetic Relationships
We constructed a phylogeny of 100 representative isolates of CC464 and associated clonal complexes using 1,643 wgMLST loci (Figure 3; Appendix Table  2). As predicted, ST5136 clustered within CC464; CC353, CC354, and CC574 isolates clustered, in that order, distal to CC464. ST5136 isolates were a single clonal MDR group closely related to the MDR isolates of ST5812 (CC464) reported in Guernsey in 2012 and now also found in England and Scotland (Figure 3). We observed the clustering of CC353, CC354, and CC574 around CC464 at the wgMLST level at which CC353 and CC354 strains had a high prevalence of ciprofloxacin resistance, whereas CC574 had a lower prevalence of ciprofloxacin resistance. Our findings concur with those reported earlier (36). Strain ST6209 (isolate no. 72, resistant only to β-lactam) clustered around ST464 isolates (nos. 73, 74, 75) that had genotypes encoding low levels of resistance (Figure 3).

Discussion
The UK broiler sector is the fourth-largest poultry meat producer in the European Union, making the United Kingdom ≈75% self-sufficient in poultry meat. The industry has benefited from the continuing consumer choice of leaner meat, reflected in the 12% rise in poultry meat sales in 2017. The widespread presence of antimicrobial resistance in Campylobacter spp. in retail poultry in the United Kingdom suggests horizontal transfer and mutational events within and between broiler farms and environmental conditions leading to proliferation of antibiotic-resistant lineages (37,38). This process is observed in ST5136, which was first detected in 2010 as an ST464 variant isolated from chicken meat (C0112) and clinical stool samples (ARI0509) from North East Scotland and Oxford (OXC5459). In 2012, ST5136 was reported from turkey meat (C0469) in North East Scotland, and in all cases from both years these isolates were MDR. ST5136 was never detected before 2010 in any of the 15,365 isolates submitted to the PubMLST Campylobacter database, but since its discovery, it has been found in >18 abattoirs across the United Kingdom. ST5136 has only ever been isolated from poultry (chicken 90%, turkey 10%, duck <1%) and never from cattle, sheep, pigs, or wild birds (39). Thus, human acquisition is probably exclusively from poultry meat.
The evolutionary ancestry of ST5136 shows that it is a member of CC464 and evolved from a heterogeneous complex of related strains, including ST464, all of which show high prevalence of antimicrobial resistance ( Figure  3). ST5136 forms a cluster within CC464, an ancestor of CC353 with CC574 and CC354 associated more distally (Figure 2).
In the PubMLST Campylobacter database, the earliest report of ST354 was from a human blood culture in 1984; in 1991 it was isolated from chicken meat in the United Kingdom (39). ST574 was isolated in Thailand in 1999 and reported in the United Kingdom from an unspecified human sample in 2000 and chicken meat in 2001 (39). The closest ancestor of ST464 is ST353, which was isolated from chicken in 1982 in the United Kingdom, and ST464, reported in 2001 from chicken meat in Northern Ireland (first report of ST464 was from Germany in 2000 from human stool) and then ST5136 reported in 2010 (39). We compiled an illustration of the acquisition of resistance markers in ST5136 from ST464 (Figure 1). In the ancestral state, most CC464 isolates harbor aac3 and then acquire ciprofloxacin resistance, tet(O/32/O) and bla OXA-61 -like. Subsequently, a mutation in the bla OXA-61 -like promoter leads to very high levels of ampicillin resistance. All these events have led to the stepwise selection of increasing drug resistance phenotypes in an intensely antimicrobial-competitive environment (Appendix Table 2). Ancestral to CC464 is CC353 and somewhat more distally CC574 and CC354, each of which shows progressively lower proportions of antimicrobial-resistant isolates. The commonality of the alleles for genes conferring resistance to the 4 different classes of antimicrobial drugs examined in this article indicates a gradual accumulation of these markers, culminating in ST5136. The most recently observed marker, gyrA 57 , is common to isolates of ST5136 and its cogenitor CC464 and leads to ciprofloxacin resistance, resulting in resistant strains that are related and are fitter than their sensitive counterparts (Figure 2; Appendix Table 2) (34). Because most ST5136 isolates show high levels of fluoroquinolone, β-lactam, aminoglycoside, and tetracycline resistance, it is likely that these related isolates evolved from a resistant ST464 ancestor.
The tet(O/32/O) 7 and aac3 17 alleles were most likely acquired previously, before the divergence of CC464 from CC353. Purifying selection of resistances could be occurring in CC464, as tet(O/32/O) 7 in this group was increasingly common compared with the ancestral strains. The tetracycline resistance gene tet(O/32/O) 7 is found on either plasmids or the chromosome in the ancestral isolates CC464, CC353, CC354, and CC574, but by the appearance of the CC464 ancestor of ST5136, it is found only on the chromosome, suggesting acquisition either via conjugation or by transformation into the last common ancestor. There is a constant flow of resistance genes between plasmids, phages, and bacteria in the farm environment and the chicken gut microbiome. Species such as Streptococcus and Enterococcus that form a part of the chicken gut microbiome often harbor promiscuous plasmids with resistance genes that can occur in Campylobacter; this exchange of genetic determinants will be a contributor to antimicrobial resistance evolution in Campylobacter (40,41). Similarly, OXA-193 dominated in isolates of CC464 that were closely ancestral to ST5136 and evolved to be more highly resistant to β-lactam antimicrobial drugs as a result of the -10 promoter mutation in the upstream region of bla  .
Superimposed onto this linear phylogeny of antimicrobial resistance in ST5136 are the genetic changes occurring in the other strains of the related lineages ( Figure 1). In Campylobacter the principal sources of genetic change are intracellular mutational events and the acquisition of extracellular genes from other isolates; these events are estimated as equally common (34,42), mutations resulting in a clonal distribution of characters in related isolates and the acquisitions in a mosaic distribution of characters across the species. These sources of genetic diversity lead to the emergence of novel variants in fitter isolates selected by the environment. Thus, for Campylobacter, there is a complex distribution of individual genetic markers when viewed in the context of the overall phylogeny of related isolates. This finding is most clearly exemplified in the example of 1 ST5136 isolate (ARI3975), which has a basal level of resistance to ampicillin and no promotor mutation; these aspects could be the result of a spontaneous back mutation or horizontal gene transfer from a sensitive strain of Campylobacter.
Resistances are propagated across the clonal population of Campylobacter; from these survival-of-the-fittest chains of events in antimicrobial drug-rich environments, such as poultry farms, may emerge more abundant lineages due to the prophylactic or metaphylactic use of drugs (43). ST464 has given rise to 2 MDR strains: ST5136 (uncA allele 1→3) and ST5812 (pgm allele 10→17), which are closely associated with each other. It is unclear whether the spread of ST5136 was better facilitated than that of ST5812 or that other genetic changes in ST5136 made it more likely than ST5812 to survive and colonize in poultry or by chance.
Dissemination of highly related Campylobacter strains throughout the poultry industry has occurred previously. In New Zealand, C. jejuni ST474 (CC48) was a dominant strain found almost exclusively in isolates from 1 poultry supplier and associated with clinical cases during 2005-2008 (44). More recently, tetracycline-and fluoroquinolone-resistant C. jejuni ST6944 (CC354, one of the ancestors of CC464) has been reported in human cases and in 3 major poultry suppliers in New Zealand (45).
The UK poultry industry has responded well to the current BPC antibiotic stewardship program with a 71% reduction in use of antimicrobial drugs from 2012 to 2016, but it is worth noting that enrofloxacin and difloxacin are authorized fluoroquinolones currently used in the chicken industry in the United Kingdom (46). A 1991 study in the Netherlands reported an increase in fluoroquinolone resistance in Campylobacter correlated with increasing use of enrofloxacin in the poultry industry and thus the transmission of fluoroquinolone-resistant Campylobacter from chickens to humans (38). In 2012, the poultry industry phased out use of the modern cephalosporins completely, but a commitment to stop the prophylactic use of fluoroquinolones in day-old chickens was not made until 2016 (46). Even then, these antimicrobial drugs may be added to the drinking water of flocks of poultry when no disease is present in most of the birds in a flock (47). The continued occurrence of resistant strains that emerge in an antimicrobial-stressed environment but retain their resistance in environments even after the cessation of antibiotic pressure has been reported previously (48). Although positive antimicrobial usage may mitigate against the creation of future MDR bacteria, preexisting MDR strains, such as C. jejuni ST5136, may never lose their resistance characteristics. Joshua Lederberg has said, "The future of humanity and microbes likely will unfold as episodes of Our Wits Versus Their Genes" (49). The evolution and rise of ST5136 indicates that bacteria can evolve by genetic adaptation to antibiotic-enriched and -deprived environments, which drives the evolution of environment-favored strains by mutation or gene transfer. It is up to us to use our wits to keep up with these changes.