Association of Campylobacter jejuni Metabolic Traits with Multilocus Sequence Types

Abstract

In this study, we describe the association of three Campylobacter jejuni metabolism-related traits, γ-glutamyl-transpeptidase (GGT), fucose permease (fucP), and secreted l-asparaginase [ansB(s)], with multilocus sequence types (STs). A total of 710 C. jejuni isolates with known STs were selected and originated from humans, poultry, bovines, and the environment. Among these isolates, we found 31.1% to produce GGT and 49.3% and 30.3% to be positive for ansB(s) and fucP, respectively. The combination of GGT production, the presence of ansB(s), and the absence of fucP was associated with ST-22, ST-586, and the ST-45 and ST-283 clonal complexes (CCs), which were the main STs and CCs found among the human and chicken isolates. The ST-21 CC was associated with the presence of fucP and was the major CC among the bovine isolates. Although the ST-61 CC was the second major CC among the bovine isolates, these isolates did not have any of the markers studied, making the role of fucP in bovine gut colonization questionable. The ST-45 CC was subdivided into three groups that were attributed solely to ST-45. One group showed a marker combination described previously, another group was found to be positive for ansB(s) only, and the third group did not have any of the markers studied. These results suggest that the host association of these markers seems to be indirect and may arise as a consequence of host-ST and -CC associations. Thus, a representative collection of STs should be tested to draw sensible conclusions in similar studies.

INTRODUCTION

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and is responsible for more than 90% of campylobacteriosis cases. The majority of C. jejuni infections are sporadic, and in many northern European countries, including Finland, a seasonal peak occurs in the summer months of June to August (29). In Finland, a total of 78 cases of campylobacteriosis per 100,000 inhabitants were registered in 2011 according to the Finnish National Infectious Diseases Registry (http://www3.ktl.fi/stat/). Case-control studies have identified a variety of risk factors, such as the consumption of raw and undercooked poultry meat (7) and contaminated water (32) and contact with farm and pet animals (1, 7), for acquiring sporadic campylobacteriosis.

Multilocus sequence typing (MLST) has been widely employed to characterize the C. jejuni population structure and for the source attributions of campylobacteriosis cases (4, 34, 39). Previously, in two longitudinal studies, we genotyped Finnish human, poultry, and bovine isolates by MLST and found host-associated sequence types (STs) and clonal complexes (CCs) (4, 22). The MLST distribution from different sources revealed that a total of 75% of all isolates studied belonged to the ST-45 CC (47%), the ST-21 CC (17%), and the ST-677 CC (11%) (4, 22). In different European countries, a similar distribution of major STs/CCs from both animal sources and human patients has been seen, with the ST-21 complex and the ST-45 complex being most frequently isolated (14, 23, 27, 33, 35).

Despite growing knowledge of C. jejuni reservoirs for human infections and the prevalence of specific STs among isolates from different sources (17), the mechanisms of colonization and virulence remain poorly understood (18). It has been proposed that explorations of the divergence in metabolic traits could yield new insights into colonization requirements and pathogenicity (9, 12, 18). Indeed, diversity in various secondary metabolic traits among isolates is evident (11, 18, 40). While the use of amino acids, including serine, proline, aspartate, and glutamic acid, by C. jejuni isolates as energy sources has been demonstrated (2, 12, 18, 24, 38), only some isolates are able to metabolize glutamine, asparagine (17), and l-fucose (28, 36). Hofreuter et al. (19) showed previously that highly pathogenic C. jejuni strain 81-176 contains the γ-glutamyl-transpeptidase (GGT) gene (ggt), which is absent in strains NCTC 11168 and RM1221. In addition, the gene encoding a fucose permease (fucP), used for the uptake of l-fucose, is present in C. jejuni strain NCTC 11168 but not in strain 81-176 or 81116 (28). Moreover, even though all sequenced C. jejuni genomes have a gene encoding l-asparaginase (ansB), certain strains (e.g., 81-176) include a secretion signal sequence in the gene [here designated ansB(s)], which is absent from other strains, such as NCTC 11168. Under certain conditions or in certain environments, these metabolic diversities can offer competitive advantages to the isolates (18, 19, 28, 36).

Previously, we demonstrated that the presence of ggt was associated with the host source of the isolates (10), and recently, Zautner et al. (40) showed that ggt and ansB(s) were strongly associated with certain MLSTs and clonal complexes (29).

The aim of this study was to associate previously MLST-typed C. jejuni isolates with GGT activity and the presence of fucP and a secretory form of ansB in order to better understand the molecular epidemiology of C. jejuni.

MATERIALS AND METHODS

Bacterial isolates and DNA isolation.

A total of 710 C. jejuni isolates with known MLSTs were selected from our collection, which included 355 isolates from domestically acquired sporadic human cases of gastroenteritis during 1996 to 2006 (4, 22); 119 isolates from bovine rectal samples isolated in 2003 (4, 15, 22); 142 isolates from poultry meat or cecal samples isolated in 1996, 2003, 2006, and 2007 (4, 16, 22, 31); and 94 isolates from environmental sources, comprising 52 isolates from natural waters and 42 isolates from wild birds and zoo animals, which were isolated in 2002, 2005, 2006, 2007, 2008, 2009, and 2010. DNA isolation was carried out as previously described (22).

GGT enzyme assay.

GGT activity was qualitatively detected according to a method described previously by Chevalier et al. (3). Briefly, bacteria were harvested after 24 h of microaerobic incubation at 37°C on nutrient blood plates and resuspended in 200 μl of water, to which 200 μl of reagent (100 mM Tris [pH 8.25], 2.9 mM l-γ-glutamyl-carboxy-3-nitro-4 anilide, and 100 mM glycylglycine) was added. The suspension was incubated at 37°C for 30 min, and the isolates were scored as GGT positive (yellow) or negative (opaque/white).

ansB(s) and fucP PCRs.

Primers used for the amplification of ansB(s) and fucP are presented in Table 1. The PCR conditions for fucP were the same as those described previously (8), and the PCR conditions for the detection of ansB(s) were as follows: 1× PCR buffer (Thermo Fisher Scientific, Vantaa, Finland), 1 U DyNAzyme DNA polymerase (Thermo Fisher Scientific), 250 μM each deoxynucleoside triphosphate (dNTP) (Thermo Fisher Scientific), 0.5 mM MgCl₂ (Sigma-Aldrich Finland Oy, Helsinki, Finland), 10 pmol each primer, and 50 ng genomic DNA. The cycling conditions were 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 45 s and a final extension step at 72°C for 10 min. The products of ansB with and without a secretion signal differed by only 40 bp; therefore, a 3% MetaPhor (Lonza, Fisher Scientific Oy) gel was used for visualization. C. jejuni strains 81-176 and NCTC 11168 were used as positive and negative controls, respectively.

Table 1.

Primer pairs used for amplification of ansB and fucP

Gene	Primer	Primer sequence (5′→3′)	Product size (bp)	Reference
ansB	ansBF	GGG GAA TGG TAA CTC CAC AA	195/236	This study
	ansB2R	CCT GCT ATC CTT CCA CCT GT
fucP	cj0486F	GAT AGA GCA TTA AAT TGG GAT G	1,200	8
	cj0486R	CCT ATA AAG CCA TAC CAA GCC

Statistical analysis.

Fisher's exact test or Pearson's chi-square test was used to assess the association of the ST or CC with GGT activity, ansB(s), and fucP when these traits were compared against the whole collection. P values of <0.05 were considered statistically significant.

Tree construction.

A dendrogram was constructed from a distance-based matrix of the allelic profiles by using the neighbor-joining method with the tree-drawing tools PHYLIP and Phylodendron (available at http://pubmlst.org/analysis/) and with the iTOL online tool (http://itol.embl.de/itol.cgi) (25, 26).

RESULTS

The 710 C. jejuni isolates studied included isolates of 165 STs, 114 of which belonged to 27 known clonal complexes. Of the 710 isolates, 31.1% produced GGT, 49.3% were positive for ansB(s), and 30.3% were positive for fucP (Table 2). Each metabolic trait was associated with certain STs and CCs (Fig. 1 and Table 2; see also Table S1 in the supplemental material).

Table 2.

Percentages of GGT-, ansB(s)-, and fucP-positive Campylobacter jejuni isolates (n = 710) from either human, poultry, bovine, or environmental sources^a

Source and CC (no. of isolates)	GGT		ansB(s)		fucP
	% positive isolates	Statistical significance	% positive isolates	Statistical significance	% positive isolates	Statistical significance
Human (355)	34.4		51.8		28.5
ST-45 CC (157)	26.5	***	29.3	***	0.6	***b
ST-21 CC (74)	0.6	***b	0.8	***b	20.6	***
ST-677 CC (35)	0.3	***b	9.6	***	0	***b
ST-22 CC (21)	3.1		5.9	***	0	**b
Others (68)	3.9		6.2	***	7.3	*
Poultry (142)	38.0	*	60.6	**	18.3	**b
ST-45 CC (67)	25.4	***	33.1	*	0	***b
ST-21 CC (18)	0	***b	0	***b	12.7	***
ST-677 CC (15)	0	***b	10.6	***	0
ST-283 CC (8)	4.9	**	5.6	*	0
Others (34)	7.7		11.3		5.6
Bovine (119)	10.1	***b	15.9	***b	57.1	***
ST-21 CC (58)	0	***b	0	***b	47.9	***
ST-61 CC (20)	0		0	*b	0	***b
ST-45 CC (13)	4.2	**	6.7	***	0	***b
ST-48 CC (6)	0		0		5.0	*
Others (22)	5.9	***	9.2	***	4.2	***b
Environmental (94)	35.1		64.9	***	21.3	*b
ST-45 CC (33)	23.4	***	33.0	***	3.2	*b
ST-1034 CC (6)	0		0	**b	1.0
ST-21 CC (4)	0		0	*b	4.3	**
UAc (31)	7.4		18.1		6.4
Others (20)	4.3		13.8		6.4
Total (710)	31.1		49.3		30.3

^aAsterisks indicate a statistically significant association (*, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

^bNegative association.

^cUA, unassigned.

Fig 1.

Neighbor-joining, unrooted, circular-dendrogram clustering of C. jejuni isolates based on sequence type (ST) profiles. The STs in the same clonal complex are shaded in the same color. Representations of STs of the four different sources, human (H), bovine (B), poultry (P), and environment (E), are indicated with red, purple, yellow, and green circles, respectively. The GGT, ansB(s), and fucP relationships with STs are indicated with white, gray, and black circles, respectively, and the size of the circle represents the proportion of an ST positive for a certain trait.

The most common combination of markers associated with an ST or a CC was GGT production, the presence of ansB(s), and the absence of fucP. However, the ST-677 complex was ansB(s) positive (P < 0.001) only and both GGT (P < 0.001) and fucP (P < 0.001) negative, whereas the ST-21 CC (P < 0.001) and the ST-48 CC (P < 0.0001) were significantly associated with the presence of fucP. An absence of all metabolic traits was found for isolates belonging to the ST-61 CC, the ST-692 CC, the ST-1034 CC, and the ST-1332 CC (Fig. 1; see also Table S1 in the supplemental material).

Within the ST-22 CC and the ST-45 CC, not all STs had the same combination of metabolic traits. All ST-22 isolates (n = 15) were positive for both GGT and ansB(s), while the ST-1947 isolates (n = 9) were positive for ansB(s) only. The ST-45 CC comprised a total of 270 isolates, 64.1% of which belonged to ST-45. Of the ST-45 isolates, 38.2% were positive for both GGT and ansB(s), 19.7% were PCR positive for ansB(s) only, and 37.6% did not have any of the markers studied.

Of the 222 GGT-positive isolates, 208 (93.7%) were also positive for ansB(s) (P < 0.001). On the other hand, more isolates were ansB(s) positive than GGT positive, and only 59.4% of the ansB(s)-positive isolates were GGT positive. Out of all of the isolates tested (n = 710), 9 (1.3%) tested positive for all metabolic traits (including all 3 of the ST-1275 CC isolates), and 156 (22%) tested negative for all metabolic traits. Eleven isolates (1.5%) tested positive for both GGT and fucP, and 22 isolates (3.1%) were positive for ansB(s) and fucP.

The poultry host was associated (P = 0.012) with the combination of GGT expression, the presence of ansB(s), and the absence of fucP. Inversely, the bovine host was negatively associated (P < 0.001) with the same combination of markers. The environmental isolates were associated only with ansB(s) (Table 2), but neither these isolates nor the human isolates were associated with a combination of markers.

DISCUSSION

In the current study, we analyzed and found associations between the production of GGT and the presence of ansB(s) and fucP with multilocus sequence types of 710 human, poultry, bovine, and environmental C. jejuni isolates. These metabolic traits were described and characterized previously in several studies, which assessed the importance of these traits for bacterial virulence, colonization, and host adaptation potential (2, 18, 28, 36). Additionally, we found that the two amino acid metabolism-associated markers, GGT and ansB(s), were either exclusively present or exclusively absent in the same STs. Instead of using a ggt PCR described previously (10), we used a qualitative enzyme assay to detect GGT enzyme activity, which is a more direct indication of the functional activity than PCR. In our previously study (10), which did not include MLST, the human and poultry hosts were associated with ggt. Here, we found that in both the human and poultry hosts, the major clonal complexes contributing to the GGT association were the ST-45 CC and the ST-283 CC. Also, these clonal complexes, as well as the ST-677 CC, were associated with ansB(s). The bovine host association with fucP resulted mostly from the fucP association with the ST-21 CC, in which 48% of our bovine isolates were found. Our present results strongly suggest that the previously observed host association with ggt is a consequence of the association of the multilocus sequence type with this metabolic trait.

In general, the STs within one CC exhibit the same metabolic trait patterns, with the exceptions of the ST-22 CC and the ST-45 CC. In our previous study (31), ST-22 was positive for GGT, whereas ST-1947 did not produce GGT, indicating that they might have evolved in different niches. In our present study, ansB(s) and fucP were uniformly distributed among the ST-22 CC and did not further divide ST-22 or ST-1947. The ST-45 CC, on the other hand, proved to be very heterogeneous. Overall, the ST-45 CC was positive for GGT and ansB(s) and negative for fucP, but ST-45 could be divided into three groups: GGT and ansB(s) positive, ansB(s) positive only, and negative for all traits. The observed heterogeneity among isolates of ST-45 was found previously in a variety of studies using Penner heat-stable serotypes (6), FlaA short-variable-region variants (5), microarrays (37), stress response analyses (13), and lipooligosaccharide locus class distributions (20, 30) and is not surprising, as ST-45 has been isolated from a wide variety of sources (21; http://pubmlst.org/campylobacter/) and seems to adapt easily to new niches. In contrast to ST-45, ST-50 (ST-21 CC) retained solely fucP. Nevertheless, ST-45 and ST-50 were found in all the sources studied here. Collectively, our results indicate that colonization in animals and infection in humans by isolates with these STs are not dependent on any combination of the three traits or one trait exclusively.

Previous studies performed in different geographic areas have shown different percentages of isolates positive for ggt, ansB(s), and fucP, depending on the MLST and the composition of the collection (11, 40). Zautner et al. (40) previously described lower percentages of isolates positive for the ggt and ansB(s) genes. However, the major CCs in their study were the ST-21, the ST-45, the ST-206, and the ST-48 CCs. All of these CCs, except for the ST-45 CC, were generally GGT and ansB(s) negative. Similarly, Gripp et al. (11) found previously that the proportion of isolates possessing the ggt and ansB(s) genes was <8%, whereas the proportion of isolates possessing fucP was 79.5%, but the major STs in their study were ST-21 and ST-50 (54.5% of their isolates). Therefore, the results of MLST marker studies are highly dependent on the ST composition and the diversity of the strain collection. This means that in MLST marker studies, an examination of a large variety and sufficient numbers of STs is necessary for a complete reflection of the homo- or heterogeneous distribution of the studied markers among the C. jejuni population.

The expression of GGT and the presence of ansB(s) were highly associated with each other, but only 59.4% of the ansB(s)-positive isolates had GGT activity. In line with findings reported previously by Muraoka and Zhang (28), we found a negative correlation between fucP and GGT and additionally between fucP and ansB(s). It is possible that during strain evolution, incompatibility and, subsequently, a loss of certain genes or gene combinations occurred. At the same time, poultry was associated with the presence of GGT and ansB(s) and the absence of fucP, whereas the bovine host was negatively associated with this combination. However, the human isolates as well as the environmental isolates were much more diverse in their trait distributions, reflecting probably a variability of sources. According to data reported previously by Barnes et al. (2) and Hofreuter et al. (18), GGT and ansB(s) could play a role in chicken gut colonization. However, it is unclear whether these amino acid metabolism markers may provide a competitive advantage in bovine gut colonization, virulence in humans, and the stress response in environmental isolates.

Our findings confirm once more that MLST is a valuable and reliable tool to characterize C. jejuni populations and to study the evolution of genetic lineages of C. jejuni as well as host preferences. Even with the addition of three metabolic markers, GGT, ansB(s), and fucP, only the ST-22 CC and the ST-45 CC were further subdivided into separate lineages. Assessing MLST results and the presence or absence of the studied metabolic traits revealed that all the sources in this study could be positive for all the traits or a combination thereof or negative for all these traits. In conclusion, the results of our study suggest that the host association of the metabolic traits GGT, ansB(s), and fucP arises as a consequence of the traits' connection with certain host-associated sequence types and clonal complexes.