Parachlamydia Acanthamoebae Research Articles

Resistance of different Chlamydia-like organisms to quinolones and mutations in the quinoline resistance-determining region of the DNA gyrase A- and topoisomerase-encoding genes

Parachlamydia acanthamoebae has been shown to be resistant to fluoroquinolones. In this study, we determined the susceptibility of Neochlamydia hartmannellae and Simkania negevensis to quinolones using amoebal co-culture in the presence of serial dilutions of ciprofloxacin. The g yrA and parC quinoline resistance-determining regions (QRDRs) were amplified and sequenced, and amino acid substitutions that may explain the observed quinolone resistance were investigated. Neochlamydia and Simkania were resistant to 16 μg/mL ciprofloxacin. Substitution at positions 83 and 70 of G yrA QRDR were present in Neochlamydia and in both Parachlamydia strains studied. For Simkania, substitutions at positions 99 and 101 in the GyrA QRDR and at positions 81 and 84 in the ParC QRDR were observed. Resistance of Parachlamydia, Neochlamydia and Simkania to quinolones is likely due to these mutations.

Chlamydiales in Guinea‐pigs and Their Zoonotic Potential

The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.

Chlamydialike Organisms and Atherosclerosis

Chlamydialike Organisms and Atherosclerosis

Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria.

Novel chlamydiae are newly recognized members of the phylum Chlamydiales that are only distantly related to the classic Chlamydiaceae, i.e., Chlamydia and Chlamydophila species. They also exhibit an obligate biphasic intracellular life cycle within eukaryote host cells. Some of these new chlamydiae are currently considered potential emerging human and/or animal pathogens. Parachlamydia acanthamoebae and Simkania negevensis are both emerging respiratory human pathogens, Waddlia chondrophila could be a novel abortigenic bovine agent, and Piscichlamydia salmonis has recently been identified as an agent of the gill epitheliocystis in the Atlantic salmon. Fritschea spp. and Rhabdochlamydia spp. seem to be confined to arthropods, but some evidence for human exposure exists. In this review, we first summarize the data supporting a pathogenic potential of the novel chlamydiae for humans and other vertebrates and the interactions that most of these chlamydiae have with free-living amoebae. We then review the diagnostic approaches to infections potentially due to the novel chlamydiae, especially focusing on the currently available PCR-based protocols, mammalian cell culture, the amoebal coculture system, and serology.

Parachlamydia acanthamoebae enters and multiplies within pneumocytes and lung fibroblasts

Parachlamydia acanthamoebae is a Chlamydia-like organism that naturally infects free-living amoebae. P. acanthamoebae is a putative emerging agent of community-acquired and inhalation pneumonia that may enter and multiply within human macrophages. However, since Parachlamydia induces their apoptosis, macrophages may not represent a perennial niche for this obligate intracellular bacterium. Therefore, we investigated whether pneumocytes and lung fibroblasts are permissive to Parachlamydia infection and might act as a replicative niche. Entry of Parachlamydia into pneumocytes (A549) and lung fibroblasts (HEL) was confirmed by confocal and electron microscopy. In A549 cells, the mean number of Parachlamydia per cell increased 7-fold from day 0 to day 7, independently of the technique used to label the bacteria. The proportion of infected A549 cells also increased over time, whereas cell viability remained unaffected by Parachlamydia infection. The sustained (3 weeks) viability of Parachlamydia when incubated in the presence of A549 cells contrasted with that observed in the absence of cells. HEL cells were also permissive to Parachlamydia infection, as we observed a 3- to 4-fold increase in the mean number of bacteria per cell. In HEL cells, Parachlamydia retained some viability for 2 weeks. These findings demonstrate that Parachlamydia is able to enter and multiply within pneumocytes and fibroblasts. The viability of both cell types was not compromised after Parachlamydia infection. We therefore conclude that these cells may remain infected for a prolonged time and may represent an intrapulmonary niche for the strictly intracellular Parachlamydia. This indirectly supports the role of Parachlamydia as an agent of pneumonia.

Ameba-associated Microorganisms and Diagnosis of Nosocomial Pneumonia

To elucidate the role of ameba-associated microorganisms (AAMs) as etiologic agents of pneumonia, we screened for Legionella spp., Parachlamydia acanthamoeba, Afipia sp., Bosea spp., Bradyrhizobium spp., Mesorhizobium amorphae, Rasbo bacterium, Azorhizobium caulinodans, Acanthamoeba polyphaga mimivirus, and conventional microorganisms in 210 pneumonia patients in intensive-care units by using culture, polymerase chain reaction, and serologic testing. These resulted in 59 diagnoses in 40 patients. AAMs and non-AAMs were implicated in 10.5% of the patients. The infectious agents were identified in 15 patients: Acanthamoeba polyphaga mimivirus, 8; Legionella pneumophila, 3; L. anisa, 1; Parachlamydia sp., 1; Bosea massiliensis, L. worsleiensis, L. quinlivanii, and L. rubrilucens, 1; and M. amorphae and R. bacterium, 1. A. polyphaga mimivirus was the fourth most common etiologic agent, with a higher seroprevalence than noted in healthy controls. This finding suggested its clinical relevance. Therefore, AAM might cause nosocomial pneumonia and should be suspected when conventional microbiologic results are negative.

‘Candidatus Protochlamydia amoebophila’, an endosymbiont of Acanthamoeba spp.

The obligately intracellular coccoid bacterium UWE25, a symbiont of Acanthamoeba spp., was previously identified as being related to chlamydiae based upon the presence of a chlamydia-like developmental cycle and its 16S rRNA gene sequence. Analysis of its complete genome sequence demonstrated that UWE25 shows many characteristic features of chlamydiae, including dependency on host-derived metabolites, composition of the cell envelope and the ability to thrive as an energy parasite within the cells of its eukaryotic host. Phylogenetic analysis of 44 ribosomal proteins further confirmed the affiliation of UWE25 to the 'Chlamydiae'. Within this phylum, UWE25 could be assigned to the family Parachlamydiaceae based on comparative analyses of the 16S rRNA, 23S rRNA and endoribonuclease P RNA genes. The distinct dissimilarities from its closest relative, Parachlamydia acanthamoebae Bn(9)(T) (7.1, 9.7 and 28.8%, respectively), observed in this analysis justify its classification in a new genus. Therefore, the name 'Candidatus Protochlamydia amoebophila' is proposed for the designation of the Acanthamoeba sp. symbiont UWE25 (=ATCC PRA-7).

Lack of microbicidal response in human macrophages infected with Parachlamydia acanthamoebae

Parachlamydia acanthamoebae is an obligate intracellular bacterium naturally infecting free-living amoebae. This potential agent of pneumonia resists destruction by human macrophages, inducing their death by apoptosis. However, the strategy used by Parachlamydia to escape the microbicidal effectors of macrophages remains unknown. In this work, we defined the effect of Parachlamydia on the cytokine secretion (measured in culture supernatants by immunoassays), on the oxidative burst (measured using a fluorogenic probe), on the production of nitric oxide (Griess assay), and on transcription of glutaredoxin, tumor necrosis factor alpha (TNF-α) and indoleamine 2,3-dioxygenase (IDO). Living Parachlamydia did not induce an oxidative burst, the secretion of cytokines such as IL-6, IL-10 and TNF-α, nor the transcription of TNF-α in macrophages. However, living Parachlamydia led to increased secretion of IL-1β and increased transcription of glutaredoxin, an anti-oxidant. The transcription of IDO, an enzyme, which catalyzes decyclization of l-tryptophan, was slightly up-regulated. Heat-inactivated Parachlamydia did not induce either an oxidative burst or the production of pro-inflammatory cytokines. In contrast to living bacteria, it had no effect on the IL-1β release, but it induced IL-10 secretion. In conclusion, after being internalized, Parachlamydia may resist the microbicidal effectors of human macrophages through not inducing oxidative burst and pro-inflammatory cytokine production.

Intracellular trafficking of Parachlamydia acanthamoebae

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.

Microorganisms Resistant to Free-Living Amoebae

Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

Emerging Chlamydial Infections

Chlamydiae are important intracellular bacterial pathogens of vertebrates. In the last years, novel members of this group have been discovered: Parachlamydia acanthamoebae and Simkania negevensis seems to be emerging respiratory human pathogens, while Waddlia chondrophila might be a new agent of bovine abortion. Various species have been showed to infect also the herpetofauna and fishes, and some novel chlamydiae are endosymbionts of arthropods. In addition, molecular studies evidenced a huge diversity of chlamydiae from both environmental and clinical samples, most of such a diversity could be formed by novel lineages of chlamydiae. Experimental studies showed that free-living amoebae may support multiplication of various chlamydiae, then could play an important role as reservoir/vector of chlamydial infections. Here we reviewed literature data concerning chlamydial infections, with a particular emphasis on the novely described chlamydial organisms.

Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis [corrected

Parachlamydia acanthamoebae is an obligately intracellular bacterium that naturally infects free-living amoebae. It is a potential human pathogen and may survive in human macrophages. We studied P. acanthamoebae entry into, and multiplication within, human monocyte-derived macrophages. After 8 h of incubation, 80% of macrophages were infected with a mean of 3.8 P. acanthamoebae organisms per cell. Electron microscopy demonstrated that parachlamydiae were in an intracellular vacuole. After infection with living organisms, the number of parachlamydiae per macrophage increased 4 times from day 0 to day 4, whereas heat-inactivated parachlamydiae were eliminated during the same period. Quantitative PCR confirmed that P. acanthamoebae replicates within macrophages. Transcriptional activity of P. acanthamoeba was detected by reverse transcription-PCR targeting the gene encoding ADP-ATP translocase (tlc). P. acanthamoebae exerted a cytopathic effect on macrophages. When macrophages were infected with living bacteria, their number decreased significantly from day 0 to day 4 due to apoptosis, as shown by annexin-V binding and electron microscopy. This study shows that P. acanthamoebae enters and multiplies within human macrophages before inducing their apoptosis.

History of the ADP/ATP-translocase-encoding gene, a parasitism gene transferred from a Chlamydiales ancestor to plants 1 billion years ago.

Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene, tlc, is found only in Rickettsiales, Chlamydiales, and plant and alga plastids. We demonstrate the presence of tlc in Parachlamydia acanthamoebae. This gene shares more similarity with the tlc1 gene of Chlamydiaceae and the tlc of plant and alga plastids than with the tlc2 gene of Chlamydiaceae. Phylogenetic analysis, including all other tlc homologs found in GenBank, showed that tlc was duplicated in a Chlamydiales ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by tlc. Plant and alga plastids acquired tlc approximately when Parachlamydiaceae and Chlamydiaceae diverged, at the eucaryotic radiation time, ca. 1 billion years ago.

New parachlamydial 16S rDNA phylotypes detected in human clinical samples

Chlamydiales are important intracellular bacterial pathogens, causing a wide variety of diseases in vertebrates, including humans. Besides the well-known species in the family Chlamydiaceae, new chlamydial organisms have recently been discovered, forming three new families: Parachlamydiaceae, Simkaniaceae and Waddliaceae. Parachlamydia acanthamoebae and Simkania negevensis are currently investigated as emerging human respiratory pathogens. Additional chlamydial lineages have been discovered by 16S rDNA-based molecular studies, and their implication in human infections is poorly known. By using a pan-chlamydia 16S rDNA PCR, we have searched for the presence of chlamydiae in 228 clinical samples that all previously had been shown to be PCR-negative for Chlamydophila pneumoniae: 170 respiratory samples, 45 atheromatic plaques and 13 peripheral blood mononuclear cell samples. Nine respiratory samples tested positive. Sequence analysis has allowed us to assign four sequences to Chlamydophila psittaci, three sequences to Chlamydophila felis, and two sequences to two novel phylotypes belonging to the Parachlamydiaceae. These latter sequences showed similarity values of more than 93% with each other and with the P. acanthamoebae sequence, thus belonging to novel, unrecognized species. In conclusion, this report showed that a variety of non- C. pneumoniae chlamydial respiratory infection is present in humans, and that new parachlamydiae distinct from P. acanthamoebae may be detected in human clinical samples. Future studies will be of interest in order to estimate the diversity of these novel chlamydiae in both clinical and environmental samples, as well as their possible clinical implication in human and animal infections.

Parachlamydiaceae: potential emerging pathogens.

Parachlamydiaceae, which naturally infect amoebae, form a sister taxon to the Chlamydiaceae on the basis of the Chlamydia-like cycle of replication and 80% to 90% homology of ribosomal RNA genes. Because intra-amoebal growth could increase the virulence of some intracellular bacteria, Parachlamydiaceae may be pathogenic. Arguments supporting a pathogenic role are that Chlamydia pneumoniae, a well-recognized agent of pneumonia, was shown to infect free-living amoebae and that another member of the Chlamydiales, Simkania negevensis, which has 88% homology with Parachlamydia acanthamoebae, has caused pneumonia in adults and acute bronchiolitis in infants. The recent identification of a 16S rRNA gene sequence of a Parachlamydiaceae from bronchoalveolar lavage is additional evidence supporting potential for pathogenicity.

Legionella-Like and Other Amoebal Pathogens as Agents of Community-Acquired Pneumonia

We tested serum specimens from three groups of patients with pneumonia by indirect immunofluorescence against Legionella-like amoebal pathogens (LLAPs) 1-7, 9, 10, 12, 13; Parachlamydia acanthamoeba strains BN 9 and Hall's coccus; and Afipia felis. We found that LLAPs play a role (albeit an infrequent one) in community-acquired pneumonia, usually as a co-pathogen but sometimes as the sole identified pathogen.

Characterization of the rnpB gene and RNase P RNA in the order Chlamydiales.

The sequence of the RNase P RNA gene (rnpB) was determined for 60 strains representing all nine species in the family Chlamydiaceae and for the related Chlamydiales species, Parachlamydia acanthamoebae and Simkania negevensis. These sequences were used to infer evolutionary relationships among the Chlamydiaceae. The analysis separated Chlamydophila and Chlamydia into two lineages, with Chlamydophila forming three distinct clusters: the Chlamydophila pneumoniae strains; the Chlamydophila pecorum strains; and a third cluster comprising the species Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae and Chlamydophila felis. The Chlamydia line of descent contained two clusters, with the Chlamydia suis strains distinctly separated from strains of Chlamydia trachomatis and Chlamydia muridarum. This analysis indicated that the rnpB sequence and structure are distinctive markers for species in the Chlamydiaceae. It was also demonstrated that the RNase P RNA derived from Chlamydia trachomatis is able to cleave a tRNA precursor in the absence of protein. These findings are discussed in relation to the structure of Chlamydia RNase P RNA.

Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms.

The current taxonomic classification of Chlamydia is based on limited phenotypic, morphologic and genetic criteria. This classification does not take into account recent analysis of the ribosomal operon or recently identified obligately intracellular organisms that have a chlamydia-like developmental cycle of replication. Neither does it provide a systematic rationale for identifying new strains. In this study, phylogenetic analyses of the 16S and 23S rRNA genes are presented with corroborating genetic and phenotypic information to show that the order Chlamydiales contains at least four distinct groups at the family level and that within the Chlamydiaceae are two distinct lineages which branch into nine separate clusters. In this report a reclassification of the order Chlamydiales and its current taxa is proposed. This proposal retains currently known strains with > 90% 16S rRNA identity in the family Chlamydiaceae and separates other chlamydia-like organisms that have 80-90% 16S rRNA relatedness to the Chlamydiaceae into new families. Chlamydiae that were previously described as 'Candidatus Parachlamydia acanthamoebae' Amann, Springer, Schönhuber, Ludwig, Schmid, Müller and Michel 1997, become members of Parachlamydiaceae fam. nov., Parachlamydia acanthamoebae gen. nov., sp. now. 'Simkania' strain Z becomes the founding member of Simkaniaceae fam. nov., Simkania negevensis gen. nov., sp. nov. The fourth group, which includes strain WSU 86-1044, was left unnamed. The Chlamydiaceae, which currently has only the genus Chlamydia, is divided into two genera, Chlamydia and Chlamydophila gen. nov. Two new species, Chlamydia muridarum sp. nov. and Chlamydia suis sp. nov., join Chlamydia trachomatis in the emended genus Chlamydia. Chlamydophila gen. nov. assimilates the current species, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia psittaci, to form Chlamydophila pecorum comb. nov., Chlamydophila pneumoniae comb. nov. and Chlamydophila psittaci comb. nov. Three new Chlamydophila species are derived from Chlamydia psittaci: Chlamydophila abortus gen. nov., sp. nov., Chlamydophila caviae gen. nov., sp. nov. and Chlamydophila felis gen. nov., sp. nov. Emended descriptions for the order Chlamydiales and for the family Chlamydiaceae are provided. These families, genera and species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes.

Molecular evidence for the existence of additional members of the order Chlamydiales.

Respiratory tract infections in man may be caused by several members of the genus Chlamydia and also by two Chlamydia-like strains, 'Simkania negevensis' (Z-agent) and 'Parachlamydia acanthamoebae' (Bng). To facilitate diagnostic procedures a PCR assay able to detect all known Chlamydiaceae sequences in one reaction was developed. For this purpose, primers were selected to amplify a fragment of the 16S rRNA gene. Characterization of the amplified fragments was done by hybridization with specific probes and by sequencing. PCR assays were carried out using DNA isolated from nose/throat specimens or from peripheral blood mononuclear cells of patients with respiratory tract infections, and from vessel wall specimens of abdominal aneurysms. Six of the 42 nose/throat swab specimens analysed yielded strong bands and one yielded a faint band. Three of these bands were identified as Chlamydia pneumoniae and one as Chlamydia trachomatis by sequencing. Analysis of the three other bands yielded two different new sequences. DNA isolated from peripheral blood mononuclear cells of one patient yielded a third new sequence. DNA isolated from peripheral blood mononuclear cells of four healthy controls was negative. One of the abdominal aneurysm specimens also yielded a strong band. Sequencing revealed a fourth new sequence. All negative controls included during specimen processing and PCR analysis remained negative. The typical secondary structure of microbial 16S genes was present in all four new sequences indicating the validity of the sequence data. All four new sequences were distinct from other bacteria and clustered together with known Chlamydiaceae sequences. Phylogenetic analysis suggested a new lineage, separating the four new sequences, 'S. negevensis' and 'P. acanthamoebae' from the genus Chlamydia with the four known chlamydial species. In conclusion, this study provides evidence for the existence of several new members of the order Chlamydiales. Since the source of the Chlamydia-like strains has not been identified and serological and/or molecular cross-reactivities may be expected, results of identification of infecting recognized organisms should be interpreted cautiously.

Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp.

The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasal mucosa of humans was analyzed by the rRNA approach. The primary structures of the 16S and 23S rRNA molecules of one strain were determined in almost full length. In situ hybridization with a horseradish peroxidase-labeled oligonucleotide probe targeted to a unique signature site undoubtedly correlated the retrieved 16S rRNA sequence to the respective intracellular parasite. This probe also hybridized with the second strain, suggesting a close relationship between the two intracellular parasites. Comparative sequence analysis demonstrated a distinct relationship to the genus Chlamydia. With 16S rRNA similarities of 86 to 87% to the hitherto-sequenced Chlamydia species, the intracellular parasites are likely not new species of this genus but representatives of another genus in the family of the Chlamydiaceae. Consequently, it is proposed to provisionally classify the endoparasite of Acanthamoeba sp. strain Bn9 as "Candidatus Parachlamydia acanthamoebae." From an epidemiological perspective, the results suggest that small amoebae could be environmental reservoirs and vectors for a variety of potentially pathogenic bacteria including members of the Chlamydiaceae.