Spread of avian pathogenic Escherichia coli ST117 O78:H4 in Nordic broiler production

Escherichia coli infections in poultry constitute a severe animal health issue and a considerable burden to farmers worldwide, in terms of decreased animal welfare and production economy [1, 2]. Disease in poultry caused by avian pathogenic E. coli (APEC) may cause a wide range of extraintestinal symptoms, collectively termed colibacillosis. APEC belong to the group of extraintestinal pathogenic E. coli (ExPEC) [3–5], that also includes the pathotypes; uropathogenic E. coli (UPEC), neonatal-meningitis E. coli (NMEC) and septicemic E. coli. All groups have been associated with disease in both humans and animals [3, 4], and it has been reported that human ExPEC strains are closely related to APEC strains, suggesting that poultry could constitute a reservoir of zoonotic APEC strains [3, 6, 7].

In general, APEC isolates from chickens constitute a genetically diverse population with numerous of different serogroups and sequence types (STs). The most commonly observed serogroups are O1, O2 and O78 [6–9], and multilocus sequence typing (MLST) has shown that STs 10, 48, 95 and 117 have been frequently observed [5, 10, 11]. Several types of virulence genes are commonly identified in APEC as well as in human ExPEC [7, 12] (Additional file 1: Table S1). These are often carried on virulence plasmids and pathogenicity islands (PAIs) [13–15].

Nordic broiler production depends on a breeding pyramid where Swedish grandparents are mainly imported from Scotland and used for breeding of parents for export to farms in the rest of the Nordic countries [16, 17]. Interestingly, previous studies indicated that extended-spectrum beta-lactamase (ESBL)-producing E. coli can be transmitted vertically from parents to offspring through the breeding pyramid [17–19]. Hence, if great grandparents are infected with virulent E. coli, they can potentially disseminate vertically to Swedish grandparents and hereafter to parents and broilers across the Nordic countries.

From 2009 until late 2014, the mortality on Danish poultry farms has on average been decreasing. Hereafter, the mortality has increased to >4.0% in 2015 and it has been suggested that colibacillosis in both parents and broilers has played a significant role [20]. In the same period similar problems with colibacillosis and increased mortality have also been observed on Finnish and Norwegian farms (Magne Hansen, Animalia, pers. comm). The aim of this study was to investigate, using whole genome sequencing, the genetic diversity and potential relatedness of APEC isolates associated with increased mortality and colibacillosis in Nordic countries.

The majority of the isolates (66/114) were sequenced to an average coverage of ≥50 fold, whereas 35/114 of the draft genomes had an average coverage of >30. The rest had an average coverage of >18, whereas seven draft genomes provided by the Danish poultry industry exhibited slightly less coverage. Assembly metrics (average coverage, N50, number of contigs and assembly size) can be found in Additional file 4: Table S4.

Serotyping and MLST

Serotype genes were in the majority of the 114 assembled genomes, identified with thresholds of ≥90% nucleotide identity, ≥90% coverage of the query and a sequence depth of >10×. However, in eight isolates (E29, E50, E52, E56, E64, E66, E69 and E91) the O-type genes were on average identified with 67% coverage of the query and ≥90% nucleotide identity, whereas no O-type genes were identified in nine isolates (Additional file 2: Table S2). The isolates showed a high diversity with a total of 33 different serotypes, and MLST analyses identified 29 different STs. The most prevalent serotype was O78:H4 observed among 43% (49/114) of the isolates, whereas 54% (62/114) were found to be of ST117 (Additional file 2: Table S2). Of all isolates, 61% (70/114) could be divided into six groups associated with the same serotype and ST (Table 1). Notably, all six groups presented in Table 1 were also closely related according to the SNP analysis (Fig. 1).

	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6	Healthy	Diseased
Isolates	47	8	5	4	3	3	15	99
Farms	34	8	4	4	2	3	15	73
Serotype	O78:H4	O53:H4	O103:H2	O18ac:H7	O149:H23	O5:H10	-	-
ST	117	117	1146	95	1163	93	-	-
fimA	+	+	+	+	+	1/3	12/15	94/99
fimC	+	+	+	+	+	+	13/15	96/99
papC								4/99
tsh				+		+		20/99
fyuA		+		3/4			1/15	28/99
iroD	+	+		+	+	+	4/15	94/99
iroN	+	+		+	+	+	3/15	93/99
irp2		+		+			1/15	30/99
iucA	+	+	+	+		+	7/15	84/99
iucD	+	+	+	+		1/3	7/15	84/99
cva		+	+	+	+	+	12/15	39/99
cvi		+	+	+	+	+	12/15	39/99
iss	+	+	2/5	+	+	+	7/15	97/99
ompA	+	+	+	+	+	+	15/15	99/99
vat	+	+		+				71/99
hlyA
ibeA				+		1/3		13/99

The table shows the virulence gene content among E coli groups of identical ST and serotype. “+” indicates presence of genes. If a virulence gene was not present among all isolates in a group, the ratio of isolates that carried the gene is presented. The number of isolates and the number of different farms they were collected from, in each group is shown. The isolates from these groups were also closely related according to the SNP analysis, and the groups are highlighted in colors in Fig. 1. Furthermore, the ratios of isolates from healthy and diseased poultry that carried virulence genes are shown

Phylogenetic analyses

The SNP calling based on all 114 isolates had a total of 145,637 variant positions identified in ~49% of the reference genome. The phylogenetic analysis revealed a large clade of 62 isolates from both diseased broilers and parents collected in Denmark, Finland and Norway. Isolates from healthy chickens were not related to this clade (Fig. 1). All isolates from this clade belonged to ST117 and 47 of these had serotype O78:H4 whereas eight had O53:H5 (Fig. 1). It was not possible to identify O-type genes in six isolates from this clade, but they all carried the H4 gene. A single isolates (E24) was of serotype O161:H4 (Fig. 1) (Additional file 2: Table S2). Minor clusters of few isolates with identical serotype and ST were present but none of them contained isolates from more than two different countries (Fig. 1).

The SNP calling based on all 83 ST117 genomes from both this study and those obtainable from the public domain at NCBI [32] had a total of 13,215 variant positions identified in ~64% of the genomes, and with 2,617 SNPs remaining after purging of recombinant regions. The analysis identified a major clade primarily consisting of 47 O78:H4 isolates from both diseased broilers and parents collected on 34 different farms in Denmark, Finland and Norway (Fig. 2). On average, the length between these 47 isolates was 23 SNPs. In four isolates (E46, E53, E54, and E72) from this clade no O-type genes were identified, whereas only one isolate in this clade (E13) belonged to a serogroup different from O78 (Fig. 2). Furthermore, strain GN02004 obtained from the RefSeq archive at NCBI (Additional file 3: Table S3) belonged to serotype O24:H4. The 47 O78:H4 isolates differed by at least 50 SNPs from the O53:H4 clade (Fig. 2).

In recent years (2014–2016), an increase in cases of colibacillosis on Nordic poultry farms has caused a raise in mortality and economic losses [20] (Magne Hansen, Animalia, pers. comm). Therefore, the genetic diversity among 114 E. coli isolates mainly collected from diseased animals on poultry farms with colibacillosis were investigated. In agreement with previous studies [5, 7, 9, 11], it was found that the poultry isolates were a genetically diverse population. However, we identified a group of 47 closely related ST117 O78:H4 isolates collected from diseased broilers and parents in multiple Nordic countries, which shared a similar genetic background (Fig. 2). In concordance, it has previously been reported that APEC isolates are widely associated with serogroup O78 and ST117 [7, 11]. These 47 isolates were not related to any of the isolates from healthy chickens (Fig. 1). On average, the distance between these 47 isolates was 23 SNPs, whereas the distance to the closest related ST117 O53:H4 isolate outside this group was 50 SNPs (Fig. 2). Additionally, all 47 ST117 O78:H4 carried an identical and unique combination of virulence genes compared to all other investigated isolates (Table 1). Thus, according to both the investigation of virulence profiles and the SNP analyses the 47 ST117 O78:H4 isolates define a distinct lineage. The isolates from this lineage carried nine genes that encode virulence factors important in the pathogenesis of avian colibacillosis (Table 1). Interestingly, O78 strains have been suggested to be the main cause of avian colisepticemia together with O1 and O2 strains [35]. The O-antigen capsule allows bacteria to avoid the host’s innate immune response and studies have shown that this LPS capsule is required during systemic infections [35, 36]. Type I fimbriae encoded by fimA/C, have been shown to be necessary for initial colonization of the respiratory system [37]. Additionally, previous studies suggest that iucA/D and iroD which encode aerobactin and salmochelin siderophores respectively, are specifically important for iron acquisition in the extraintestinal environment of chickens [38]. As in this study, the virulence factor increased serum survival encoded by iss, was previously identified in APEC strains [7, 12] as well as the vacuolating autotransporter toxin Vat, but their exact role in the pathogenesis needs to be further elucidated [15, 39]. It should also be noticed that some of the virulence genes (fimA/C, ompA) were found among almost all analyzed isolates, both from healthy and diseased birds, which indicates that they are not only involved in avian colibacillosis. Additionally, a wide range of other virulence factors have also been suggested to be associated with APEC [40]. None of the investigated virulence genes (Table 1) were found exclusively among the O78:H4 cluster and it may therefore be suggested that other, yet undefined virulence mechanisms were partly responsible for the high virulence of this strain. Thus, a comparative study to reveal more precisely why the O78:H4 ST117 lineage was considerably associated with increased mortality among Nordic broilers and breeders from 2014–2016 could be interesting to carry out.

In the Nordic countries, all poultry farms receive their parents from Swedish hatcheries where grandparents are imported from Scotland [16, 17]. It was not possible to verify how many Swedish parent flocks that the birds included in this study originated from, which could have further revealed the extent of the colibacillosis issues observed on Nordic poultry farms. Finding highly similar isolates in broilers and parents from distantly located farms that share one common source for parent animals strongly support a vertical dissemination of ST117 O78:H4 isolates from grandparents and great grandparent. Vertical transmission of pathogenic E. coli has previously been observed in other studies [17–19]. Unfortunately, E. coli isolates from diseased grandparents or great grandparents were not available to further confirm this hypothesis. It could have been interesting to investigate samples from parents and their corresponding offspring but a parent flock can be origin to several different broiler flocks. Thus, it is not possible to collect samples directly from parent/offspring pairs [16]. Instead, an in vivo infection study of parents and their offspring could be carried out.

The 47 O78:H4 ST117 isolates carried various types of virulence genes (Table 1) that previously have been identified in both APEC and human UPEC isolates. Additionally, they were closely related to the ST117 O24:H4 E. coli strain GN02004 from NCBI (Fig. 2), which previously has been collected from human body fluids. (Additional file 3: Table S3). Therefore, it could be speculated whether the origin of these colibacillosis cases could have been the introduction of human UPEC to grandparents or great grandparents in the upper parts of the breeding pyramid. Strain GN02004 was sampled in USA but we could not obtain further information regarding possible dissemination via travel activities to Europe. Thus, diseased chickens could potentially constitute a zoonotic risk to e.g., farmers or other people who get in contact with them [3, 7]. Additionally, E. coli strains have been found to end up in the broiler chicken meat [17].

Since the increase in the occurrence of colibacillosis has a considerable impact on animal welfare and production economy, it is of great importance to obtain more in-depth knowledge regarding APEC and colibacillosis and to develop vaccines that possibly could provide immunization of the poultry. Interestingly, E. coli strain E44 [21] collected from a diseased parent bird, was here shown to be a part of the major ST117 O78:H4 linage. In 2015, strain E44 was selected for a Danish autovaccine program due to its suggested relation to the increase in colibacillosis on Danish poultry farms. However, the efficiency of the program remains to be evaluated.

Genomic investigation of APEC isolates collected from diseased chickens on Nordic poultry farms revealed the presence of a predominant lineage of ST117 O78:H4 isolates. The analyses indicated that the ST117 O78:H4 strains have been transmitted vertically through the broiler breeding pyramid and contributed considerably to the increase of colibacillosis cases observed on Nordic poultry farms from 2014 to 2016.