Helicobacter ibis sp. nov., isolated from faecal droppings of black-faced ibis (Theristicus melanopis)

Description of Helicobacter ibis sp. nov. isolated from faecal droppings of 1 black-faced ibis (Theristicus melanopis) 2 Mónica Lopez-Cantillo1, Boris Vidal-Veuthey1, Armin Mella1, Rafael R. de la Haba2 and 3 Luis Collado1 4 5 Author affiliations:1Institute of Biochemistry and Microbiology, Faculty of Sciences, 6 Universidad Austral de Chile, Valdivia, Chile. 2Department of Microbiology and 7 Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain. 8 9 Corresponding author: Luis Collado; Phone: 56-63-2293848, E-mail: lu[email protected] 10 11 Keywords: Helicobacter, faeces, wild bird, Chile. 12 13 Repositories: 14 The following gene sequences have been deposited in GenBank/EMBL/DDBJ: 16S rRNA 15 gene sequences of strains A82T and WB-40 (ON950425 and ON950426); and hsp60 gene 16 sequences of strains A82T and WB-40 (OQ116579 and OQ116580). The accession 17 numbers for the draft genome sequence data of strains H. ibis A82T and WB-40 are 18 JAQHXR000000000 and JAQHXS000000000, respectively. 19 20 21 22 23 Abstract 24 As part of a larger study on Epsilonproteobacteria carried by wild birds in the city of 25 Valdivia (southern Chile), two curved rod-shaped Gram-stain-negative strains (A82T and 26 WB-40) were recovered from faecal samples and subjected to a taxonomic study. 27 Results of a genus-specific PCR showed that these isolates belonged to the genus 28 Helicobacter. Further identification by 16S rRNA and hsp60 (60 kDa heat-shock protein) 29 gene sequence analysis revealed that they formed a separate phylogenetic clade, 30 different from other known Helicobacter species with “H. burdigaliensis” CNRCH 31 2005/566HT and H. valdiviensis WBE14T being the most closely related species. This was 32 confirmed by core-genome phylogeny as well as digital DNA-DNA hybridization (dDDH) 33 and average nucleotide identity (ANI) analyses between the genome of strains A82T and 34 WB-40 and the rest of Helicobacter species. The draft genome sequence of A82T and 35 WB-40, obtained by Illumina NextSeq 2000 sequencing, consisted of ∼1.6 Mb with a 36 G+C content of 31.9-32.0 mol%. 37 The results obtained from the phylogenetic and genomic characterisation, together with 38 their different morphological and biochemical features, revealed that these two strains 39 represent a novel species, for which we propose the name Helicobacter ibis sp. nov. with 40 A82T (=LMG 32718T= CCCT 22.04T) as the type strain. 41 42 Introduction 43 The genus Helicobacter contains 51 validly published species names 44 (https://lpsn.dsmz.de/genus/helicobacter) which can be divided into two main groups: 45 gastric (GH) and enterohepatic Helicobacter (EHH) species. The latter includes more 46 than 60 % of the species of the genus, and in recent years the EHH group has been linked 47 to human diseases such as acute gastroenteritis, inflammatory bowel and hepatobiliar 48 diseases, as well as extra-intestinal infections [1]. 49 50 The EHH species have been isolated from a wide range of wild and domestic animals [2]. 51 However, the pathogenic significance of these bacteria is uncertain as they have been 52 detected both in healthy animals and in those with symptoms of intestinal or liver 53 disease [2]. Wild birds are an important reservoir for EHH, with H. pametensis [3], H. 54 anseris, H. brantae [4], H. canadensis [5], and H. valdiviensis [6] having been isolated 55 from faecal samples of these hosts. Additionally, very recently two new species from 56 birds were described in Turkey, “H. anatolicus” and H. kayseriensis [7]. Some of these 57 species have also been linked to human infection. Indeed H. canadensis was originally 58 isolated from diarrheic faeces [8], and H. valdiviensis has also been detected in human 59 diarrheal stool specimens, alongside systemic infection [9]. Evidently, wild bird 60 droppings can be a source of spreading EHH and other campylobacteria, as they 61 contaminate parks, lakes, and urban areas, which could pose a risk to human health [10]. 62 In the framework of a study developed in the city of Valdivia (southern Chile), on the 63 prevalence and diversity of Epsilonproteobacteria in wild bird dropping samples, two 64 bacterial strains (A82T and WB-40) were subjected to phylogenetic analysis using the 16S 65 rRNA and hsp60 (60 kDa heat-shock protein) gene sequences, and were suspected of 66 belonging to a not-yet described Helicobacter species. Whole genome-based analyses 67 and phenotypic characterisation were consequently performed to determine their 68 taxonomic positions. Based on our results, we propose and describe these strains as 69 representing a novel species within the genus Helicobacter. 70 71 Isolation and identification 72 Strains A82T and WB-40 were isolated in November 2020 and January 2022, respectively. 73 The samples analysed were from faecal droppings excreted by birds in public urban 74 green spaces in Valdivia. Faecal samples were collected using sterile cotton-tipped 75 swabs that were immediately placed in 9 ml of Bolton broth (Oxoid). Incubation was 76 performed at 37 °C for 48 h under micro-aerobic conditions (Anaerocult® C, Merck 77 Millipore). After enrichment, 100 μL of incubated broth was inoculated on a modified 78 charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid) plate, and incubated at 42 79 °C. Meanwhile 400 μL of broth was placed on the surface of a Millipore membrane filter 80 (pore size 0.45 μm) on Columbia Blood Agar Base (Oxoid) supplemented with 5 % sheep 81 blood (CBA) and was allowed to filter passively under room conditions for approx. 30 82 min. The filter was later removed with sterile forceps and discarded, while the plate was 83 incubated at 37 °C for 48 h under the above-mentioned conditions. In cases where no 84 growth was observed, incubation was extended for an additional 5–7 days [6]. Finally, 85 along with several known Campylobacter and Helicobacter species, three isolates 86 suspected of belonging to a new Helicobacter species were obtained from the 270 wild 87 bird faecal droppings analysed. These isolates were genotyping by Enterobacterial 88 repetitive intergenic consensus PCR [11] resulting in two strains with different 89 genotypes (A82T isolated on CBA and mCCDA, and WB-40 isolated only on CBA) (data 90 not shown). In an attempt to identify the origin of the samples, faecal DNA was extracted 91 using the Stool DNA Kit (Omega Biotek) and the mitochondrial COI (cytochrome c 92 oxidase subunit 1) gene was amplified and sequenced using the universal primers BirdF1 93 and BirdR1, and alternative reverse primer BirdR2 [12]. Only one of the two bird faecal 94 samples from which the strains under study were isolated generated a good quality 95 sequence (GenBank accession number OP236466), corresponding to strain WB-40. A 96 BLASTN search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) identified the bird sequence as 97 belonging to the black-faced ibis (Theristicus melanopis) with a 100 % identity score. It 98 is one of the most abundant bird species observed at the sampling site. 99 100 Bacterial DNA was extracted from A82T and WB-40 colonies using the Wizard Genomic 101 DNA Purification kit (Promega), in accordance with the manufacturer’s instructions. 102 Genus-identification was carried out by Helicobacter genus-specific PCR [13]. For 103 identification at species level, the 16S rRNA and hsp60 genes were amplified and 104 sequenced as described by Vandamme et al. [14] and Hill et al. [15], respectively. DNA 105 sequences were then compared directly with the NCBI non-redundant nucleotide 106 database using BLASTN, and top hit sequences were retrieved for further comparisons. 107 Sequence alignment was performed with ClustalW [16] and phylogenetic trees were 108 reconstructed using the neighbour-joining method [17] as implemented using MEGA X 109 software [18], with Kimura’s two-parameter evolutionary distance model [19]. The 110 stability of the groupings was estimated by bootstrap analysis based on 500 re- 111 samplings. The phylogenetic tree based on 16S rRNA gene sequences (Fig. 1) clearly 112 showed that strains A82T and WB-40 can be affiliated to the genus Helicobacter, most 113 closely related to “H. burdigaliensis” CNRCH 2005/566HT (97.10 % similarity) and H. 114 valdiviensis WBE14T (97.02 % similarity). Given that the two new isolates were closely 115 related, forming a single cluster, it was hypothesized that they may represent a single 116 species. This taxonomic situation of the novel strains was also confirmed by the hsp60 - 117 based phylogenetic tree (Fig. 2). 118 It is important to highlight the taxonomic similarity of the new taxon (putatively 119 comprised of strains A82T and WB-40) to H. valdiviensis, considering that both species 120 were isolated in the same geographic area and from the same type of reservoir. 121 122 Genome analysis 123 Genomic DNA of strains A82T and WB-40 was extracted using an Easy-DNA™ gDNA 124 Purification Kit (Invitrogen), and further quantified by spectrophotometry (NanoQuant 125 – Infinite M200, Tecan) and fluorometry (Qubit 3.0 fluorometer). Genomic libraries were 126 prepared using the Illumina DNA Prep kit and IDT 10 bp UDI indices, and then sequenced 127 on an Illumina NextSeq 2000 device at the Microbial Genomics Sequencing Center 128 (MIGS, Pittsburgh, PA, USA). Reads were quality checked with FastQC v.0.11.9 [20], 129 filtered with Trimmomatic v.0.39 [21], and finally assembled using SPAdes v3.13.1 [22] 130 (option --careful). Assembly quality was assessed using Quast v5.0.2 [23]. Completeness 131 and contamination of the genomes was estimated using CheckM v1.0.6 at default 132 settings [24]. The genome sequences were annotated using Prokka v1.14.6 [25]. 133 Sequence reads and assemblies were submitted to the NCBI database as BioProject 134 PRJNA913774 with SRA accession numbers SRX18781885 and SRX18781886, and 135 GenBank accession numbers JAQHXR000000000 and JAQHXS000000000 for strains A82T 136 and WB-40, respectively. The draft genome sequences of strain A82T and WB-40 were 137 approx. 1,593,022 bp and 1,638,157 bp long, respectively, with a DNA G+C content of 138 31.9 mol% and 32.0 mol%, respectively, which is in line with values reported for the 139 genus Helicobacter [26]. The chromosome of the type strain A82T is predicted to contain 140 approx. 1,655 coding sequences (CDS). 141 142 Available genome sequences from all type strains of members of the genus Helicobacter 143 were recovered from public databases and further used to carry out a phylogenomic 144 analysis. Orthologous genes were determined using an all-versus-all BLASTP comparison 145 among the translated CDS features of the annotated genomes under study, as previously 146 described [27]. Translated single-copy core gene sequences were then individually 147 aligned with Muscle v.3.8.31 [28] (options -maxiters 1 -diags) and concatenated into a 148 super-protein alignment, which was further utilized to infer the phylogenomic tree by 149 means of the approximately maximum-likelihood algorithm, as implemented in 150 FastTreeMP v.2.1.8 [29]. Branch support values were estimated using the Shimodaira- 151 Hasegawa test [30]. The new strains A82T and WB-40 formed a separate lineage, 152 different from all other Helicobacter spp. but closely related to “H. burdigaliensis” and 153 H. valdiviensis (Fig. 3), confirming the results obtained from the initial single marker- 154 based phylogenetic analyses (Figs. 1 and 2). 155 156 Overall Genome Relatedness Indexes (OGRI) were computed for the genomes of isolates 157 A82T and WB-40 and those of the type strains of the Helicobacter species. Specifically, 158 Orthologous Average Nucleotide Identity (OrthoANI) was determined using the 159 OrthoANIu tool [31] and the digital DNA-DNA hybridization (dDDH) was inferred by 160 means of the Genome-to-Genome Distance Calculator (GGDC) (formula 2) [32]. The 161 OrthoANI and dDDH values obtained for all the analysed genomes revealed that the two 162 new isolated strains belonged to the same species (OrthoANI: 99.6 %, dDDH: 95.9%), 163 while being different from the other Helicobacter species described to date (OrthoANI 164 ≤73.6 % and dDDH ≤36.5 %) (Fig. S1). 165 166 Phenotypic characterisation 167 The new Helicobacter strains were biochemically, physiologically, and morphologically 168 characterised according to the recommendations of On et al. [33]. All tests were 169 performed in two time-independent assays with freshly prepared media. Growth of 170 both strains was examined at 25 °C, 37 °C and 42 °C under microaerobic conditions 171 (using Anaerocult® C, Merck Millipore), and at 37 °C under aerobic and anaerobic 172 conditions (using AnaeroGen, Oxoid) for 48 to 72 h on CBA. An oxidase test was carried 173 out using Bactident Oxidase strips (Merck) and catalase activity was evaluated by adding 174 a 3 % H2O2 solution and observing bubble production within 5 s. Indoxyl acetate 175 hydrolysis was determined as previously described by Mills and Gherna [34]. The 176 following biochemical analyses were performed using the API-Campy identification 177 system (bioMérieux) as per the manufacturers’ instructions: urease activity, reduction 178 of nitrate, esterase activity, hydrolysis of hippurate, γ-glutamyl transferase activity, 179 reduction of triphenyl tetrazolium chloride, alkaline phosphatase activity, production of 180 H2S, assimilation of glucose, and pyrrolidonyl-, L-arginine-, and L-aspartate arylamidase 181 activity. In addition, nitrate reduction was also tested using plate method of Cook [35] 182 and γ-glutamyl transferase activity was confirmed using DIATABS™ Diagnostic Tablets 183 for Bacterial Identification (Rosco). Tolerance to 1 % glycine, 1.5 % NaCl and 2 % NaCl 184 was determined as previously described [36]. The motility and susceptibility to 185 cephalothin (30 μg, Oxoid) and nalidixic acid (30 μg, Oxoid) were also determined, 186 according to the Minimal Standards recommended for the families Campylobacteraceae 187 and Helicobacteraceae [33]. All test were performed using the type and reference 188 strains Helicobacter valdiviensis (CECT 8410T); H. apodemus RAT 1 (=CCUG 73261); H. 189 canicola CAD 722 (=CCUG 73259); Campylobacter jejuni (DSM 4688T); C. coli (DSM 190 4689T); C. lari (DSM 11375T); C. subantarcticus (LMG 24377T); C. insulaenigrae (LMG 191 22716T); C. volucris (LMG 24380T) and Escherichia coli (ATCC 25922) as controls. 192 Morphological characteristics of the strain A82T were observed using a transmission 193 electron microscope (LIBRA 120 PLUS, Zeiss) after negative staining with 2 % uranyl 194 acetate for 30 s. This strain showed a spiral shape with a pair of unsheathed bipolar 195 flagella (Fig. 4). Based on the phenotypic characterisation, the new strains can be 196 affiliated to the Helicobacter genus, since they comply with the typical characteristics of 197 the genus described both in its original description [37] and in its subsequent 198 emendation [38], such as to be Gram negative curved rods, nonsporeforming, motile by 199 flagella, microaerophilic, with oxidase activity, chemoorganotrophs and asaccharolytic. 200 Table 1 shows the most important phenotypic characteristics differentiating the novel 201 strains from the phylogenetically closest Helicobacter species. An extended comparison 202 with all Helicobacter taxa can be found in Supplementary Table S1. 203 204 In summary, the phylogenetic, genomic, and phenotypic results obtained demonstrate 205 that the two isolates recovered from wild bird faecal samples represent a novel taxon, 206 distinct from other currently known Helicobacter species. The name Helicobacter ibis sp. 207 nov. is proposed, with A82T (=LMG 32718T= CCCT 22.04T) as the type strain. 208 209 Description of Helicobacter ibis sp. nov. 210 Helicobacter ibis (i'bis. L. gen. n. ibis, of an ibis, indicating the source of a new 211 Helicobacter strain). 212 213 31. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to 353 calculate average nucleotide identity. Antonie Van Leeuwenhoek 2017; 110:1281- 354 1286. https://doi.org/10.1007/s10482-017-0844-4 355 32. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. 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Neighbour-joining tree based on 16S rRNA gene sequences showing the 403 phylogenetic position of Helicobacter ibis sp. nov. within the genus Helicobacter. 404 Bootstrap values (≥70 %) based on 500 replicates are shown at the nodes. Bar, 0.01 405 substitutions per nucleotide position. 406 Fig. 2. Neighbour-joining tree based on hsp60 gene sequences showing the phylogenetic 407 position of Helicobacter ibis sp. nov. within the genus Helicobacter. Bootstrap values (≥70 408 %) based on 500 replicates are shown at the nodes. Bar, 0.05 substitutions per 409 nucleotide position. 410 Fig. 3. Approximately maximum-likelihood phylogenomic tree based on 318 411 concatenated core protein sequences of members of the genus Helicobacter showing the 412 position of the new isolated strains. Ultrafast bootstrap values ≥70 % are indicated above 413 the branches. Bar, 0.1 substitutions per nucleotide position. 414 Fig. 4. Transmission electron micrograph of strain A82T; bar, 2 μm. 415 Table 416 Table 1. Phenotypic characteristics differentiating Helicobacter ibis sp. nov. from other 417 related species of the genus Helicobacter. 418 *Goodwin et al. [37] reported that some strains lose their urease activity after being repeatedly subcultured. §Weak reaction detected (in two out of seven strains). †One out of six strains was resistant. ‡The tests were assessed in plates with 32 mg L-1 of the respective antibiotic. Table 1. Phenotypic characteristics differentiating Helicobacter ibis sp. nov. from other related species of the genus Helicobacter. Helicobacter species: 1, H. ibis sp. nov. (Data from this study); 2, H. pylori (Data from Goodwin et al. [37]; Eaton et al. [39]); 3, H. pullorum (Stanley et al. [40]); 4, H. rodentium (Shen et al. [41]); 5, H. mesocricetorum (Simmons et al. [42]); 6, H. ganmani (Robertson et al. [43]); 7, H. canadensis (Fox et al. [8]); 8, H. valdiviensis (Collado et al. [6]); 9, H. monodelphidis (Shen et al. [44]); 10, H. turcicus (Aydin et al. [11]); 11, H. colisuis (Gruntar et al. [45]). +, 100 % of strains positive; -, 100 % of strain negative; (+), 70–94 % of strains positive; (-), 7–33 % of strains positive; ±, 42-66 % of strains positive; W, weak growth; S, sensitive; R, resistant; V, variable; ND, not determined; M, monopolar; B, bipolar. Characteristic 1 2 3 4 5 6 7 8 9 10 11 Catalase + + (+) + + -§ + + + - + Nitrate reduction + - + + (+) + ± - (-) - + Urease - +* - - - - - ± - - - Alkaline phosphatase + + - - + - - - + + + γ-Glutamyl transpeptidase + + ND - - ND - - + - V Indoxyl acetate hydrolysis - - - - ND - + + - + - Growth at 42 °C (microaerobic) + - + (+) + - + + - + + Growth at 37 °C (anaerobic) W - - + - + - - - + + Growth on 1 % glycine - - - + - - + + - - - Susceptibility to (30 µg per disc): Nalidixic acid S R S R S† S‡ R V R R R Cephalothin R S R R R R‡ R R R R R No. of flagella 2 4-8 1 2 2 2 1-2 1 7-14 2 2 Sheathed flagella - + - - - - - - - - - Flagellar arrangement B M M B B B B M B B B