Unity in variety--the pan-genome of the Chlamydiae.

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model.

Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.

Simkania negevensis in bronchoalveolar lavage of lung transplant recipients: a possible association with acute rejection.

BACKGROUND: Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been associated with community acquired pneumonia and acute exacerbation of chronic obstructive pulmonary disease. The prevalence and pathogenic potential of S. negevensis is not known in lung transplant recipients. METHODS: In this multicenter study comparative analysis of bronchoalveolar lavage (BAL) in lung transplants (Tx) and kidney Tx, immunocompromised and nasopharyngeal (NP) washes of immunocompetent patients was done. The BAL specimens were tested by nested polymerase chain reaction (PCR) for C. pneumoniae and S. negevensis. Selected S. negevensis positive PCR cases were confirmed by culture. RESULTS: In the initial 41 BAL samples S. negevensis was detected in 97.5% (40/41) of lung transplant recipients as compared to 14.1% (1/7) in other organ transplant recipients (P<0.0001). In the sequential samples of 19 lung transplant recipients, 59% (24/41) had concomitant positive PCR and rejection as compared to 30% (3/10) who had negative PCR but had rejection (P=0.16). S. negevensis infection had hazard ratio of 3.29 (95% CI: 0.73-14.76; P=0.11) for developing acute rejection. CONCLUSION: S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.

Infection with Simkania negevensis in Brooklyn, New York.

BACKGROUND: Simkania negevensis is a Chlamydia-like intracellular organism that is prevalent in populations from a wide range of geographic areas. The role of the organism in respiratory disease in the United States is unknown. OBJECTIVE: To study the association between infection with S. negevensis and bronchiolitis, pneumonia or asthma in Brooklyn, New York. MATERIALS AND METHODS: Pediatric and adult inpatients/outpatients with bronchiolitis, pneumonia or asthma were recruited, and a similar number of healthy control subjects were enrolled. Nasopharyngeal swabs were obtained for culture of S. negevensis and Chlamydia pneumoniae and polymerase chain reaction detection of S. negevensis. Sera were obtained for measurement of antibodies to S. negevensis and C. pneumoniae. RESULTS: One hundred eighty-eight patients and 110 healthy control subjects were enrolled. S. negevensis serologic assays were positive for 18% of patients, compared with 29% of control subjects (P = 0.09). S. negevensis DNA was detected by PCR for 17% of case subjects and 23% of control subjects (P = 0.25). S. negevensis was isolated by culture for 1 patient with bronchiolitis. C. pneumoniae IgG and S. negevensis IgG were found to increase with increasing age, ie, 14%, 50% and 78% (C. pneumoniae) and 13%, 17% and 33% (S. negevensis) for subjects 0-18 months, 18 months-18 years and older than 18 years of age, respectively. CONCLUSION: S. negevensis was not a significant respiratory pathogen in Brooklyn, NY, during the period of the study.

Infections with the chlamydia-like microorganism Simkania negevensis, a possible emerging pathogen.

Although evidence for the existence of numerous chlamydia-like microorganisms has been discovered in both environmental samples and clinical specimens, very few have been grown in vitro, and little is known of their pathogenic potential. Of all such organisms, Simkania negevensis is probably the most extensively studied. This review summarizes current knowledge about this intracellular bacterium, focusing especially on human infections.

Pneumonic vs nonpneumonic acute exacerbations of COPD.

STUDY OBJECTIVE: To describe and compare the background, clinical manifestations, disease course, and infectious etiologies of pneumonic acute exacerbations (PNAE) vs nonpneumonic acute exacerbations (NPAE) of COPD. DESIGN: A prospective, observational study. SETTING: A tertiary university medical center in southern Israel. PATIENTS: Twenty-three hospitalizations for PNAE and 217 hospitalizations for NPAE were included in the study. Paired sera were obtained for each of the hospitalizations and were tested serologically for 12 pathogens. Only a significant change in antibody titers or levels was considered diagnostic. RESULTS: No significant differences were found between the two groups for any of the parameters related to COPD or comorbidity. The clinical type of the exacerbation was not significantly different between the groups. Compared to NPAE, patients with PNAE had lower PO(2) values at hospital admission (p = 0.004) but higher rates of abrupt onset (p = 0.005), ICU admissions (p = 0.006), invasive mechanical ventilation (p = 0.01), mortality (p = 0.007), and longer hospital stay (p = 0.001). In 22 PNAE hospitalizations (96%) and in 153 NPAE hospitalizations (71%), at least one infectious etiology was identified (p = 0.001). Mixed infection was found in 13 patients with PNAE (59%) and in 59 patients with NPAE (39%; not significant [NS]). Viral etiology was identified in 18 patients with PNAE (78%) compared with 99 patients with NPAE (46%; p = 0.003). Pneumococcal etiology was found in 10 patients with PNAE (43%) and in 38 patients with NPAE (18%; p = 0.006). An atypical etiology was identified in 8 patients with PNAE (35%) and 64 patients with NPAE (30%; NS). CONCLUSIONS: Community-acquired pneumonia is common among patients hospitalized for an acute exacerbation of COPD and is generally manifested by more severe clinical and laboratory parameters. In PNAE, compared to NPAE, viral and pneumococcal etiologies are more common, but the rate of atypical pathogens is similar. The therapeutic significance of these findings should be investigated further.

A comparative study of the etiology of adult upper and lower respiratory tract infections in the community.

Lower respiratory tract infection and upper respiratory tract infection (URTI) are very common, but the etiology is not diagnosed in routine practice. The objective of this study was to determine and compare the frequency distribution of the various infectious etiologies for these diseases. One hundred seventy five adults in the community with febrile LRTI and 75 with febrile URTI were included in a purely serologically based prospective study. Paired sera were obtained for each of the patients and were tested by EIA or immunofluorescence methods to identify 14 different pathogens. Only a significant change in antibody titers between the paired sera was considered diagnostic. At least one infectious etiology was identified in 167 patients (67%). In the LRTI group, infection with at least one of 7 respiratory viruses was found in 88 patients (50%). One of the atypical pathogens was found in 40 patients (23%), of these Legionella spp. in 19 (11%) and Mycoplasma pneumoniae in 18 (10%). A bacterial etiology was found in 19 patients (11%), of these Streptococcus pneumoniae in 8 (5%) and beta-hemolytic streptococci group A in 5 (3%). The frequency distribution of etiologies in the URTI group was not significantly different from the LRTI group, except for M. pneumoniae that was identified in only one patient with URTI (p = 0.015). More than one etiologic agent was found in 42 (17%) of the patients. LRTI is caused by a broad spectrum of etiologies, with respiratory viruses predominating and a moderate, but significant, prevalence of atypical pathogens. The frequency distribution of etiologies for URTI is similar to LRTI. In a significant proportion of patients with URTI and LRTI there is serologic evidence of infection with more than one pathogen. The justification and benefit of distinguishing between URTI and LRTI in routine clinical work is doubtful. When a decision is reached to treat RTI patients with an antibiotic, it is logical to use a macrolide or tetracycline.

Versatility of Simkania negevensis infection in vitro and induction of host cell inflammatory cytokine response.

OBJECTIVE: Simkania negevensis (Sn) is an intracellular microorganism belonging to the family Simkaniaceae in the order Chlamydiales and has been associated with respiratory tract infections in infants and adults. The aim of this study was to analyze the outcome of Sn infection in different cell types. METHODS: The results of Sn infection were examined by infectivity assays, PCR and EM. The cellular response to infection was evaluated by following the synthesis of mRNA for inflammatory cytokines and cytokine secretion. RESULTS: Infections could be active, with production of progeny and cytopathic effects (CPE); persistent, induced by iron depletion or in minimally permissive cell types, with small numbers of infectious progeny; or cryptic, with no CPE or infectious progeny, but with Sn DNA detected. EM showed an abundance of EB and multiplying RB in active infection, small inclusions with mainly single RB particles in persistent infection, and aberrant inclusions in cryptic infection. We report reversion to active infection of iron-induced or spontaneous persistence; attempts to "cure" persistence with antibiotic treatment resulted in the absence of infectivity but not in the eradication of Sn DNA. CONCLUSION: Sn infections are versatile and induce a host cell inflammatory response, which may be relevant to potential Sn pathologies in vivo.

Domestic water supplies as a possible source of infection with Simkania.

OBJECTIVES: Simkania negevensis, a Chlamydia-like microorganism that has been associated with respiratory infections in children and adults, can multiply within free-living amoebae; moreover, it has been detected in domestic water supplies. The aim of this study was to determine whether there is similarity between Simkania organisms found in water and those detected in clinical samples. METHODS: PCR, membrane immunoassay for the detection of Simkania antigen, and isolation in cell culture were used for the detection of S. negevensis in nasopharyngeal wash samples (NPW) of 34 children with pneumonia and in domestic water samples from their homes. Sequencing of PCR amplicons was used for comparison of Simkania strains isolated from clinical samples and from water samples. RESULTS: In 26 cases (76%) both NPW and water were positive, and partial 16S rDNA sequences suggested that they may be the same organisms. CONCLUSION: Simkania found in domestic water supplies may be transmitted to children .

The growth cycle of Simkania negevensis.

Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'Simkania Z', belongs to the order Chlamydiales, assigned to the family Simkaniaceae: The purpose of this study was to investigate the production of Simkania negevensis progeny in infected cells in comparison with the well-documented Chlamydiaceae developmental cycle. It was found that replicating Simkania negevensis in Vero cells resembled the reticulate bodies of all known chlamydial species: in electron micrographs they were reticulated, homogeneously staining, and often caught in the process of binary division. These replicative forms were found in low abundance shortly after infection, but by 3 days post-infection they were the most prevalent particles in host cells. Electron-dense forms of Simkania negevensis began to appear on the third day post-infection, but quantitatively did not account for the high titre of infectivity in extracts from these host cells. These had both electron-dense and electron-lucent areas, a characteristic seen only in a few chlamydial species. Simkania negevensis infectivity did not appreciably change during the ensuing 12 days required for host cell lysis, despite an eightfold increase in the proportion of electron-dense bacteria over this time. The emergence of electron-dense bodies, increase in infectivity and host-cell lysis were not synchronized developmental events. This is a novel finding in Chlamydiales spp. and suggests that Simkania negevensis will provide new perspectives in the mechanisms of chlamydial intracellular growth.

High rate of Simkania negevensis among Canadian inuit infants hospitalized with lower respiratory tract infections.

To determine the prevalence of Simkania negevensis in causing pulmonary infections in children, nasopharyngeal washes were obtained from 22 infants hospitalized with acute bronchiolitis in the Baffin Island, Canada. 14 (63.6%) were positive for S. negevensis. Mixed infections with other respiratory viruses were common. All patients recovered without specific antibiotic treatment. Even though a high prevalence of S. negevensis was found, this organism may potentially well be an opportunistic agent rather than a true pathogen.

Evidence for the presence of Simkania negevensis in drinking water and in reclaimed wastewater in Israel.

Simkania negevensis is a recently discovered chlamydia-like intracellular microorganism which has been associated with bronchiolitis in infants and with community-acquired pneumonia in adults; a high seroprevalence of antibodies to the microorganism has been found in various population groups. S. negevensis can be grown in various cell lines as well as in free-living amoebae such as Acanthamoeba polyphaga. In this study, evidence for the existence of Simkania or Simkania-like microorganisms in drinking water and in reclaimed wastewater is presented for the first time. Detection of the microorganism was made possible by the development of a specific and sensitive filter membrane immunoassay and was confirmed by PCR detection of microbial DNA in the water samples. The common presence of S. negevensis in water sources together with the high seroprevalence of antibodies to it and early age of acquisition of infection may implicate water as a source of infection. The possible significance of this finding for public health and for municipal water testing and treatment needs to be further examined.

Infectious aetiologies in elderly patients hospitalised with non-pneumonic lower respiratory tract infection.

OBJECTIVE: to identify the infectious aetiologies of non-pneumonic lower respiratory tract infections in hospitalised elderly patients, and to characterise the patients in terms of demographic, clinical and therapeutic variables. DESIGN: a prospective, non-interventional, purely serologically based diagnostic study. SETTING: a tertiary university hospital in southern Israel. SUBJECTS: 133 elderly patients hospitalised for non-pneumonic lower respiratory tract infections. METHODS: paired sera were obtained for each of the hospitalisations and were tested using immunofluorescence or enzyme immunoassay methods to identify 13 different pathogens. Only significant changes in antibody titers or levels between the paired sera were considered diagnostic. RESULTS: at least one infectious aetiology was identified in 77 patients (58%). At least one of seven viral aetiologies was identified in 52 patients (39%). A bacterial aetiology was identified in 27 patients (20%) including Streptococcus pneumoniae in 24 (18%). An atypical bacterium was found in 27 patients (20%) including Mycoplasma pneumoniae in 15 (11%) and Legionella spp. in nine (7%). More than one aetiology was found in 23 patients (17%). One hundred and twenty nine patients (96%) suffered from serious chronic co-morbidity. One hundred and twenty one patients received antibiotics during their hospitalisation, 106 (80%) with a beta-lactam and 42 (31%) with another antibiotic. CONCLUSIONS: non-pneumonic lower respiratory tract infection is caused in hospitalised elderly patients by a broad spectrum of aetiological agents, primarily respiratory viruses with a significant, though lesser, prevalence of classical and atypical bacteria. Despite this distribution of aetiologies, most patients are treated with beta-lactam antibiotics. The indication for antibiotic therapy in these patients and the choice of antibiotic preparation should be addressed in further studies.

Atypical pathogen infection in adults with acute exacerbation of bronchial asthma.

In a serologically based prospective study, acute infections with four atypical pathogens were determined in 100 adults hospitalized for acute exacerbation of bronchial asthma, and compared with the corresponding rate in a matched control group. Paired sera were tested using immunofluorescence or enzyme immunoassay methods to establish the serologic diagnosis. In 18 patients (18%), there was evidence of acute infection with Mycoplasma pneumoniae, compared with 3% in the control group (p = 0.0006). In 10 of these patients there was evidence of infection with at least one additional pathogen, a respiratory virus in 7. There was no significant difference between the study groups in the rates of acute infection by Chlamydia pneumoniae (8% in the hospitalized patients versus 6% in the control subjects), Legionella spp. (5 versus 3%, respectively), or Coxiella burnettii (no patients in either group). We conclude that of these four atypical pathogens, only infection with M. pneumoniae is associated with hospitalization for acute exacerbation of bronchial asthma. In most of these M. pneumoniae patients there is evidence of infection with a respiratory virus as well. The pathophysiologic and therapeutic significance of these findings should be tested in further studies specifically designed to address these questions.