Chlamydia Species Research Articles

Chlamydiosis in Animals

The Chlamydiaceae family consists of Gram-negative, obligate intracellular bacteria that replicate within the cells of a diverse range of hosts. These hosts include domesticated animals such as cats, dogs, and livestock, as well as wildlife like koalas and birds, exotic species such as reptiles and amphibians, and humans. Chlamydial infection can result in various clinical signs, including respiratory diseases, reproductive failures, ocular pathologies, and enteritis, though the infected organism may remain asymptomatic. In recent years, chlamydial nomenclature has undergone several revisions due to the wide range of hosts, the frequent discovery of novel strains, and the reclassification of existing ones. Given this and the clinical significance of these infections, ranging from asymptomatic to fatal, an updated review is essential. This article outlines key characteristics of Chlamydia species and provides an updated overview of their nomenclature, offering a concise reference for future research on chlamydial diseases.

Insights into the Two Most Common Cancers of Primitive Gut-Derived Structures and Their Microbial Connections.

The gastrointestinal and respiratory systems are closely linked in different ways, including from the embryological, anatomical, cellular, and physiological angles. The highest number (and various types) of microorganisms live in the large intestine/colon, and constitute the normal microbiota in healthy people. Adverse alterations of the microbiota or dysbiosis can lead to chronic inflammation. If this detrimental condition persists, a sequence of pathological events can occur, such as inflammatory bowel disease, dysplasia or premalignant changes, and finally, cancer. One of the most commonly identified bacteria in both inflammatory bowel disease and colon cancer is Escherichia coli. On the other hand, patients with inflammatory bowel disease are at risk of several other diseases-both intestinal (such as malnutrition and intestinal obstruction, besides cancer) and extraintestinal (such as arthritis, bronchiectasis, and cancer risk). Cancers of the lung and colon are the two most common malignancies occurring worldwide (except for female breast cancer). Like the bacterial role in colon cancer, many studies have shown a link between chronic Chlamydia pneumoniae infection and lung cancer. However, in colon cancer, genotoxic colibactin-producing E. coli belonging to the B2 phylogroup may promote tumorigenesis. Furthermore, E. coli is believed to play an important role in the dissemination of cancer cells from the primary colonic site. Currently, seven enteric pathogenic E. coli subtypes have been described. Conversely, three Chlamydiae can cause infections in humans (C. trachomatis may increase the risk of cervical and ovarian cancers). Nonetheless, striking genomic plasticity and genetic modifications allow E. coli to constantly adjust to the surrounding environment. Consequently, E. coli becomes resistant to antibiotics and difficult to manage. To solve this problem, scientists are thinking of utilizing suitable lytic bacteriophages (viruses that infect and kill bacteria). Several bacteriophages of E. coli and Chlamydia species are being evaluated for this purpose.

Epidemiology, Transmission Mode, and Pathogenesis of Chlamydia pecorum Infection in Koalas (Phascolarctos cinereus): An Overview.

Chlamydial infections pose a significant threat to koala populations. Chlamydia pecorum (C. pecorum) remains the major chlamydial species affecting koala health, both in the wild and in captivity, and chlamydial infections are considered important factors affecting the long-term survival of koalas. A clear understanding of chlamydial infections, including the epidemiology, transmission mode, pathogenesis, immune response, control, and prevention thereof, is essential for improving the management of chlamydial infections in koalas. In this study, we discuss the important advances made in our understanding of C. pecorum infection in koalas, focusing on the epidemiology of chlamydial infections, and the transmission, pathogenesis, immune response, and control strategies for chlamydial infection, with the aim of improving koala health and achieving effective conservation strategies.

The Occurrence of Chlamydia felis in Cats and Dogs in Hungary.

The World Health Organization (WHO) estimates that many human infections are zoonoses, creating a worldwide public health challenge. Among Chlamydia species, Chlamydia felis is the leading cause of conjunctivitis in cats and is a prominent zoonotic species. This study aimed to determine the occurrence and risk of chlamydiosis in cats and dogs in Szeged, Hungary, and surrounding areas. The total nucleic acids from conjunctival swab samples of symptomatic and asymptomatic animals were extracted using an automated nucleic acid extraction system. After that, DNA was amplified by pan-chlamydia PCR. Bacterial and fungal cultures were also performed to detect other microorganisms. Of the 93 animals, 32 (34.4%) were positive for pan-chlamydia PCR. The positivity rates were 33.3% (26/78) in cats and 40.0% (6/15) in dogs. Furthermore, the positivity rates were 37.2% (16/43) in the cat shelter, 42.4% (14/33) in the veterinary clinic, and 11.7% (2/17) in household pets. In total, 103 species were identified through culture-based examinations, including 97 (94.2%) bacterial and 6 fungal (5.8%) species. From both human and animal health perspectives, it is essential to have a detailed understanding of the circumstances of chlamydiosis, given the global impact of zoonotic diseases.

Chlamydia in pigs: intriguing bacteria associated with sub-clinical carriage and clinical disease, and with zoonotic potential.

Chlamydiae are bacteria that are intriguing and important at the same time. The genus Chlamydia encompasses many species of obligate intracellular organisms: they can multiply only inside the cells of their host organism. Many, perhaps most animals have their own specifically adapted chlamydial species. In humans, the clinically most relevant species is Chlamydia trachomatis, which has particular importance as an agent of sexually transmitted disease. Pigs are the natural host of Chlamydia suis but may also carry Chlamydia abortus and Chlamydia pecorum. C. abortus and possibly C. suis have anthropozoonotic potential, which makes them interesting to human medicine, but all three species bring a substantial burden of disease to pigs. The recent availability of genomic sequence comparisons suggests adaptation of chlamydial species to their respective hosts. In cell biological terms, many aspects of all the species seem similar but non-identical: the bacteria mostly replicate within epithelial cells; they are taken up by the host cell in an endosome that they customize to generate a cytosolic vacuole; they have to evade cellular defences and have to organize nutrient transport to the vacuole; finally, they have to organize their release to be able to infect the next cell or the next host. What appears to be very difficult and challenging to achieve, is in fact a greatly successful style of parasitism. I will here attempt to cover some of the aspects of the infection biology of Chlamydia, from cell biology to immune defence, epidemiology and possibilities of prevention. I will discuss the pig as a host species and the species known to infect pigs but will in particular draw on the more detailed knowledge that we have on species that infect especially humans.

Tracking Chlamydia – Host interactions and antichlamydial activity in Caenorhabditis elegans

The fading efficacy of antibiotics is a growing global health concern due to its life-threatening consequences and increased healthcare costs. Non-genetic mechanisms of antimicrobial resistance, such as those employed by Chlamydia pneumoniae and Chlamydia trachomatis, complicate treatment as these bacteria can enter a non-replicative, persistent state under stress, evading antibiotics and linking to inflammatory conditions. Understanding chlamydial persistence at the molecular level is challenging, and new models for studying Chlamydia-host interactions in vivo are urgently needed. Caenorhabditis elegans offers an alternative given its immune system and numerous orthologues of human genes. This study established C. elegans as an in vivo model for chlamydial infection. Both Chlamydia species reduced the worm's lifespan, their DNA being detectable at three- and six-days post-infection. Azithromycin at its MIC (25 nM) failed to prevent the infection-induced lifespan reduction, indicating a persister phenotype. In contrast, the methanolic extract of Schisandra chinensis berries showed anti-chlamydial activity both in vitro (in THP-1 macrophages) and in vivo, significantly extending the lifespan of infected C. elegans and reducing the bacterial load. Moreover, S. chinensis increased the transcriptional activity of SKN-1 in the worms, but was unable to impact the bacterial load or lifespan in a sek-1 defective C. elegans strain. In summary, this study validated C. elegans as a chlamydial infection model and showcased S. chinensis berries' in vivo anti-chlamydial potential, possibly through SEK/SKN-1 signaling modulation.

Chlamydia abortus in brown pelicans (Pelecanus occidentalis) from the gulf of California, Mexico

The brown pelican (Pelecanus occidentalis californicus) is a migratory subspecies that nests on the islands of the gulf of California, in the Pacific Northwest. The objective of this study was to identify Chlamydia species in brown pelican samples obtained in 2016, 2017, and 2021. Samples were taken from the cloacae and choanae of 86 brown pelicans in specimens ranging from three to seven weeks of age. The samples were obtained with sterile swabs and transferred to an SPG growth medium. The DNA was extracted to carry out a real-time PCR that detects the gene of the 235 rRNA subunit of the Chlamydiaceae family. In addition, real-time PCR directed to the ompA gene was performed to determine the Chlamydia species. Of the 86 sampled pelicans, four were positive for the family Chlamydiaceae and Chlamydia abortus. This is the first report of C. abortus in brown pelicans from the gulf of California, Mexico.

A TriAdj-Adjuvanted Chlamydia trachomatis CPAF Protein Vaccine Is Highly Immunogenic in Pigs.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.

Challenges in Chlamydial Serology: Insights from a Belgian and a Dutch Population Cohort.

Serology routinely serves as a diagnostic tool to confirm Chlamydia infections in humans. Particularly in delayed settings, such as post-outbreak scenarios where the acute phase of infection has subsided, serology is invaluable. Multiple studies, nonetheless, indicate deficiencies in specificity and sensitivity of current chlamydial antibody detection assays. Incorporation of multiple antigens per target is known to improve the accuracy of chlamydial serological assays. We, therefore, used the recomLine test (Mikrogen diagnostics) on serological samples of two cohorts, as it is the only commercially available test allowing detection of antibodies against three human pathogenic Chlamydia species (C. trachomatis, C. pneumoniae and C. psittaci) using multiple antigens per target. The first cohort (n = 156; samples collected between 2008 and 2022 during a C. trachomatis screening initiative) comprised women from the Netherlands (NL) with past exposure to C. trachomatis, while the second cohort (n = 44; samples collected in 2018 in a health examination survey) consisted of Belgian citizens (BE) with occupational or recreational exposure to chickens, representing a risk population for C. psittaci. The test indicated a statistically equivalent C. pneumoniae seroprevalence in both cohorts (39.10% in NL and 34.09% in BE; p = 0.337). As expected C. trachomatis seroprevalence was significantly higher (p < 0.001) in the Dutch cohort (48.72%), as compared to the Belgian cohort (4.55%). Lastly, C. psittaci seroprevalence did not significantly differ between the two groups (2.27% in BE and 1.92% in NL; p = 0.633), even though a higher prevalence was expected for the Belgian cohort. This prompts us to question whether the Belgian cohort truly constituted a C. psittaci risk population or whether the recomLine test is susceptible to cross-reaction of species-specific antibodies, thereby increasing C. psittaci prevalence in the Dutch cohort. We advocate for the development of affordable, highly sensitive antibody detection assays that can effectively distinguish between chlamydial species, addressing the increasing demand for enhanced serological testing methodologies.

Investigation of Chlamydia pecorum in livestock from Switzerland reveals a high degree of diversity in bovine strains

Chlamydia pecorum is a widespread veterinary chlamydial species causing endemic infections in livestock, such as ruminants and pigs, globally. However, there is limited contemporary knowledge on infecting strain diversity in various hosts. This study aimed to evaluate the genetic diversity of C. pecorum strains infecting Swiss livestock through C. pecorum genotyping and phylogenetic analyses in comparison to the global population, while also assessing chlamydial strains for plasmid carriage. A total of 263 C. pecorum positive samples from clinically healthy ruminant and pig herds (Bovines = 216, sheep = 25, pigs = 14) as well as placentae from eight C. pecorum positive ruminant abortion cases from other Swiss herds were investigated. The ompA and Multi-Locus sequence typing revealed novel C. pecorum genotypes, and bovine strains exhibited considerable genetic diversity, contrasting with lower diversity in sheep and pig strains. C. pecorum plasmid was detected in 100.0% of sheep (41/41) and pig (255/255) samples, and in 69.4% of bovine samples (150/216). In contrast, no plasmid was detected in the eight C. pecorum-positive ruminant abortion cases either representing plasmid-less strains or possibly escaping PCR detection due to autolysis of the placenta. This study supports the genetic diversity of C. pecorum strains, particularly in bovines, and identifies novel sequence types in Swiss livestock.

Diversity and genetic characterization of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis)

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22–1.23 Mbp and a plasmid with a size of 7.7–8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a β lineage. A chromosome sequence had approximately 1,018–1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I–III) with high bootstrapping statistic support. Interestingly, isolate 12–01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics.

Restriction and evasion: a review of IFNγ-mediated cell-autonomous defense pathways during genital Chlamydia infection.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection (STI) in the USA. As an STI, C. trachomatis infections can cause inflammatory damage to the female reproductive tract and downstream sequelae including infertility. No vaccine currently exists to C. trachomatis, which evades sterilizing immune responses in its human host. A better understanding of this evasion will greatly benefit the production of anti-Chlamydia therapeutics and vaccination strategies. This minireview will discuss a single branch of the immune system, which activates in response to genital Chlamydia infection: so-called "cell-autonomous immunity" activated by the cytokine interferon-gamma. We will also discuss the mechanisms by which human and mouse-adapted Chlamydia species evade cell-autonomous immune responses in their native hosts. This minireview will examine five pathways of host defense and their evasion: (i) depletion of tryptophan and other nutrients, (ii) immunity-related GTPase-mediated defense, (iii) production of nitric oxide, (iv) IFNγ-induced cell death, and (v) RNF213-mediated destruction of inclusions.

Belgian Cross-Sectional Epidemiological Study on Zoonotic Avian Chlamydia spp. in Chickens.

Chlamydia psittaci, Chlamydia gallinacea, and Chlamydia abortus are the most common Chlamydia spp. in chickens and have a confirmed or suggested zoonotic potential. No recent data are available on their prevalence and impact in the Belgian chicken industry or in the recreational chicken branch. Therefore, a cross-sectional epidemiological study was executed where samples were collected from both factory-farmed and backyard chickens. More specifically, pharyngeal chicken swabs were obtained from 20 chicken farms, 5 chicken abattoirs, and 38 different backyard locations and were analyzed using species-specific Polymerase Chain Reactions (PCRs) for the presence of the three avian Chlamydia spp. To investigate their zoonotic potential, samples were simultaneously collected from 54 backyard chicken caretakes and 37 professional chicken caretakers or abattoir employees and analyzed using species-specific PCRs as well. This study confirmed the presence of DNA of all three Chlamydia species in both the chicken industry and backyard settings. Chlamydia psittaci was the most prevalent in the industry chickens (11.0%), whereas Chlamydia gallinacea was the dominant species in the backyard chickens (14.5%). Chlamydia abortus infections were more common in the commercial chickens (9.0%) compared to the backyard chickens (2.6%). The DNA of all three species was also detected in humans (3.9% Chlamydia psittaci, 2.9% Chlamydia gallinacea, and 1.0% Chlamydia abortus).

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as β-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Chlamydiae as symbionts of photosynthetic dinoflagellates.

Chlamydiae are ubiquitous intracellular bacteria and infect a wide diversity of eukaryotes, including mammals. However, chlamydiae have never been reported to infect photosynthetic organisms. Here, we describe a novel chlamydial genus and species, Candidatus Algichlamydia australiensis, capable of infecting the photosynthetic dinoflagellate Cladocopium sp. (originally isolated from a scleractinian coral). Algichlamydia australiensis was confirmed to be intracellular by fluorescence in situ hybridization and confocal laser scanning microscopy and temporally stable at the population level by monitoring its relative abundance across four weeks of host growth. Using a combination of short- and long-read sequencing, we recovered a high-quality (completeness 91.73% and contamination 0.27%) metagenome-assembled genome of A. australiensis. Phylogenetic analyses show that this chlamydial taxon represents a new genus and species within the Simkaniaceae family. Algichlamydia australiensis possesses all the hallmark genes for chlamydiae-host interactions, including a complete type III secretion system. In addition, a type IV secretion system is encoded on a plasmid and has previously been observed for only three other chlamydial species. Twenty orthologous groups of genes are unique to A. australiensis, one of which is structurally similar to a protein known from Cyanobacteria and Archaeplastida involved in thylakoid biogenesis and maintenance, hinting at potential chlamydiae interactions with the chloroplasts of Cladocopium cells. Our study shows that chlamydiae infect dinoflagellate symbionts of cnidarians, the first photosynthetic organism reported to harbor chlamydiae, thereby expanding the breadth of chlamydial hosts and providing a new contribution to the discussion around the role of chlamydiae in the establishment of the primary plastid.

Longitudinal study of Chlamydia pecorum in a healthy Swiss cattle population

Chlamydia pecorum is a globally endemic livestock pathogen but prevalence data from Switzerland has so far been limited. The present longitudinal study aimed to get an insight into the C. pecorum prevalence in Swiss cattle and investigated infection dynamics. The study population consisted of a bovine herd (n = 308) located on a farm in the north-eastern part of Switzerland. The herd comprised dairy cows, beef cattle and calves all sampled up to five times over a one-year period. At each sampling timepoint, rectal and conjunctival swabs were collected resulting in 782 samples per sampled area (total n = 1564). Chlamydiaceae screening was performed initially, followed by C. pecorum-specific real-time qPCR on all samples. For C. pecorum-positive samples, bacterial loads were determined. In this study, C. pecorum was the only chlamydial species found. Animal prevalences were determined to be 5.2–11.4%, 38.1–61.5% and 55–100% in dairy cows, beef cattle and calves, respectively. In all categories, the number of C. pecorum-positive samples was higher in conjunctival (n = 151) compared to rectal samples (n = 65), however, the average rectal load was higher. At a younger age, the chlamydial prevalence and the mean bacterial loads were significantly higher. Of all sampled bovines, only 9.4% (29/308) were high shedders (number of copies per μl &gt;1,000). Calves, which tested positive multiple times, either failed to eliminate the pathogen between sampling timepoints or were reinfected, whereas dairy cows were mostly only positive at one timepoint. In conclusion, C. pecorum was found in healthy Swiss cattle. Our observations suggested that infection takes place at an early age and immunity might develop over time. Although the gastrointestinal tract is supposed to be the main infection site, C. pecorum was not present in rectal samples from dairy cows.

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone.

Chlamydia, comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis, a close phylogenetic relative of human C. trachomatis, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin (inv), located within a chromosomal region between the rRNA operon (rrn) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis. These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication (ori) and selection markers but lacked the native chlamydial plasmids or its ori. We first recovered allele-replacement mutants using a vector that targets the tryptophan (trp) operon of C. suis. The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori. Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene.IMPORTANCEThe obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

Potential zoonotic agents in sea turtles: Chlamydia species and Listeria monocytogenes

Abstract Background Sea turtles are well recognized sentinels of marine environment as well as carrier of pathogens and contaminants. Chlamydial species have been described in reptiles, but scarce data regarding sea turtles are available in literature (Pace et al., 2022). Hitherto, only one case of listeriosis has been described in marine reptiles (Di Renzo et al., 2022). This study reports the detection of Chlamydia species and Listeria monocytogenes in loggerhead sea turtles (Caretta caretta), suggesting the role of these animals as carriers of potential zoonotic agents. Methods In 2022, a total of 11 sea turtles found stranded along Emilia-Romagna coast, Northwestern Adriatic Sea, were subjected to post mortem investigation. Cloacal swabs were collected from 10 of the investigated animals and screened for Chlamydiaceae family by qPCR targeting 23S rRNA gene, followed by confirmation via 16S rRNA fragment sequencing. For positive samples, Chlamydia species identification was performed by species-specific qPCR for C. abortus, C. pneumoniae, C. pecorum, C. psittaci. One subject showed internal organs disseminated with multiple nodular lesions that were sampled for histopathology analyses, alongside microbiological examination. The cultured isolates were identified by MALDI-TOF (MALDI Biotyper; Bruker Daltonics Inc). Results In six out of 10 cloacal swabs Chlamydiaceae DNA was demonstrated by qPCR and confirmed by sequencing of 16S rRNA, but species identification was not achieved. In the turtle with nodular lesions, histopathology revealed the presence of granulomas: the Ziehl-Neelsen stain was negative for acid-fast organisms. L. monocytogenes was isolated from heart and liver and listeriosis was diagnosed. Conclusions The results highlight how sea turtles can be carriers, symptomatic or not, of pathogens relevant for public health. Given the zoonotic potential of these microorganisms, particular precautions are recommended especially for professionals handling these animals. Key messages • Sea turtles can play an active role as carriers of possibly pathogenetic microorganisms. • Further studies of molecular identification of Chlamydia spp. from sea turtles are needed in atypical strains.

The Fish Pathogen "Candidatus Clavichlamydia salmonicola"-A Missing Link in the Evolution of Chlamydial Pathogens of Humans.

Chlamydiae like Chlamydia trachomatis and Chlamydia psittaci are well-known human and animal pathogens. Yet, the chlamydiae are a much larger group of evolutionary ancient obligate intracellular bacteria that includes predominantly symbionts of protists and diverse animals. This makes them ideal model organisms to study evolutionary transitions from symbionts in microbial eukaryotes to pathogens of humans. To this end, comparative genome analysis has served as an important tool. Genome sequence data for many chlamydial lineages are, however, still lacking, hampering our understanding of their evolutionary history. Here, we determined the first high-quality draft genome sequence of the fish pathogen "Candidatus Clavichlamydia salmonicola", representing a separate genus within the human and animal pathogenic Chlamydiaceae. The "Ca. Clavichlamydia salmonicola" genome harbors genes that so far have been exclusively found in Chlamydia species suggesting that basic mechanisms important for the interaction with chordate hosts have evolved stepwise in the history of chlamydiae. Thus, the genome sequence of "Ca. Clavichlamydia salmonicola" allows to constrain candidate genes to further understand the evolution of chlamydial virulence mechanisms required to infect mammals.