Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

Chlamydia suis is a swine pathogen that also causes zoonotic infections. Many strains contain a tet(C) cassette conferring tetracycline resistance originally acquired by horizontal gene transfer from a Gram-negative donor. As we and others have described the capacity for Chlamydiaceae to exchange DNA by homologous recombination, tet(C)-containing C. suis strains represent a potential source for antibiotic-resistance spread within and between Chlamydia species. Here, we examine the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by plaque assay. C. suis strain Rogers132, which lacks IS200/IS605 transposases, was the most efficient donor, producing three confirmed recombinants of 56 (5.4%) clones with a minimal inhibitory concentration (MIC) of ~8µg/mL. Resistance was stable when recombinants were grown initially in tetracycline at twice the MIC of S45 (0.032 µg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two sites in different recombinants: a 55kb exchange between nrqF and pckG, and a 175kb exchange between kdsA and cysQ. Neither site was associated with repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydia species that infect humans, a matter of public health importance.

Introduction

Chlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression.

Methods

We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site.

Results

The 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorectal" clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains.

Discussion

The absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae. Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis. In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk (Buteo jamaicensis), is an outlying strain with admixture of C. abortus, C. psittaci, and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus. Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages.

Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydiaceae species that infect humans, a matter of public health importance.

Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae. Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis. In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk (Buteo jamaicensis), is an outlying strain with admixture of C. abortus, C. psittaci, and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus. Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages.

Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydiaceae species that infect humans, a matter of public health importance.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

[This corrects the article DOI: 10.3389/fmicb.2023.1154664.].

Chlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots.