Complete genomes of two Chlamydia psittaci isolated from ducks and pigeons in China

TL;DR

This paper presents the complete genome sequences of two Chlamydia psittaci strains from ducks and pigeons in China, highlighting their genetic differences.

Contribution

The study provides new complete genome sequences of two C. psittaci strains with distinct ompA genotypes from avian hosts in China.

Findings

The duck strain CPS-QD/LS belongs to ompA genotype A.

The pigeon strain CPS-BY/JY belongs to ompA genotype B.

Abstract

Chlamydia psittaci is an important zoonotic pathogen that can transmit from avian to human. Herein, we report complete genome sequences of two C. psittaci strains isolated from ducks and pigeons in China. The duck strain CPS-QD/LS was identified as ompA genotype A, while the pigeon strain CPS-BY/JY was identified as genotype B.

Figures

Clustering analysis of Chlamydia psittaci ompA genes.

Tables

TABLE 1. Sample isolation data and genome characteristics

	CPS-QD/LS		CPS-BY/JY
Parameters	Chromosome	Plasmid	Chromosome	Plasmid
GenBank accession no.	CP103952.1	CP103953.1	CP184490.1	CP184489.1
Clean data (bp)	1,087,063,417 (Nanopore)		2,137,810,282 (Nanopore)
	929,758,353 (Illumina)		1,829,539,148 (Illumina)
Read length for Nanopore (bp)	174,286 (N50 9.04 kb)		473,993 (N50 5.55 kb)
Read length for PacBio (bp)	6,339,706		12,381,778
Contig length (bp)	1,149,432	7,553	1,168,334	7,553
G + C content (%)	39.08	32.95	39.05	32.82
Sequencing depth (×)	392.5 (Nanopore)		486.89 (Nanopore)
	468.04 (Illumina)		305.58 (Illumina)
No. of pseudogenes	79	0	88	0
No. of tRNAs	39	0	39	0
No. of rRNAs	3	0	3	0
No. of tmRNAs	3	0	1	0
No. of misc_RNA	3	0	2	0
SRA accession no.	SRR33065692		SRR33068449

Full text

ANNOUNCEMENT

Chlamydia psittaci is a unique zoonotic intracellular bacterial pathogen that can infect avian species, mammals, and humans (1, 2). Based on ompA gene sequences, the C. psittaci isolates can be classified into nine classical (A to F, E/B, M56, and WC) and several atypical genotypes (3). Previous studies have shown that C. psittaci infections in avian species are common in China. However, few genome data are available for analysis of its genetic diversity and host specificity. During routine detection of Chlamydia species in our laboratory, genomic DNA was isolated from pure cultures of C. psittaci using the TIANamp Genomic DNA Kit (TIANGEN, Beijing) (4). We isolated two C. psittaci strains from real-time PCR-positive duck lung tissue and pigeon cloacal swab samples by inoculating the samples into specific-pathogen-free embryonated chicken eggs (5). The isolates obtained were confirmed by real-time PCR (6) and were named as CPS-QD/LS (duck strain) and CPS-BY/JY (pigeon strain) according to their host origin region. The isolates were then inoculated into the L929 cell line for propagation and purification for genome sequencing.

Purified genome DNA was prepared for sequencing using a combination of Illumina NovaSeq 6000 and Nanopore PromethION platforms. The Illumina library was prepared using the TruSeq DNA Library Prep Kit (Illumina, San Diego, USA) and amplified within the flow cell on an Illumina cBOT instrument for cluster generation (NovaSeq 6000 PE Cluster Kit, Illumina). The clustered flow cell was loaded onto a NovaSeq 6000 sequencer (Illumina) for paired-end sequencing. The purified libraries were loaded onto primed R9.4 Spot-On flow cells and sequenced on a PacBio sequencer (Pacific Biosciences, Menlo Park, USA). Raw reads were filtered with fastp (v.0.23.2) (7), yielding 929,758,353 and 1,829,539,148 bp of clean data for strains CPS-QD/LS and CPS-BY/JY, respectively. In parallel, libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, USA) and sequenced on the PromethION platform (Oxford Nanopore Technologies, Oxford, UK) (8). Raw reads were quality-filtered (Q ≥ 7) and length-filtered (≥1,600 bp), generating 1,087,063,417 and 2,137,810,282 bp of pass reads for CPS-QD/LS and CPS-BY/JY, respectively, with N50 values of 9.04 and 5.55 kb. Hybrid genome assembly of Illumina and Nanopore reads was performed using SOAPdenovo (v.2) (9). The repeat sequence analysis was performed using the software RepeatMasker (4.1.2-p1) (10). Circularization of contigs and re-orientation to the dnaA start were assessed and corrected using BLAST. Gene prediction was carried out with Prokka (v.1.14.6) (11). Promoter regions were predicted using PromPredict (v.V1) (12), and transmembrane proteins were identified with TMHMM (v.2.0c) (13). All software was run with default parameters.

The final genomes of C. psittaci were complete, and both genomes comprised a circular chromosome and a plasmid, with GenBank accession numbers CP103952.1, CP103953.1, CP184490.1, and CP184489.1. The genomes were annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (v.6.2/v.6.9). The genome characteristics are summarized in Table 1. Sequence analysis of the ompA gene of the two obtained isolates showed that duck strain CPS-QD/LS was identical to that of a duck isolate, SZ15, and two human isolates in China (14), which could be classified to the ompA genotype A group (Fig. 1), while the ompA gene sequence of CPS-BY/JY matched the C. psittaci strains of the typical genotype B group (15), as shown in Fig. 1. These data may improve our understanding of the genetic diversity and host specificity of this Chlamydia species.

Clustering analysis of Chlamydia psittaci ompA genes.