Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Simkania negevensis in Crohn's Disease.

BACKGROUND: Simkania negevensis is an obligate intracellular Gram-negative bacterium (family Simkaniaceae, order Chlamydiales) that has been isolated from domestic and mains water supplies, is able to infect human macrophages, and can induce an inflammatory response in the host. METHODS: From June to December 2016, in a single-center observational study, colonic Crohn's disease patients and controls (subjects undergoing screening for colorectal cancer) underwent blood tests to identify serum-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) to S. negevensis and a colonoscopy with biopsies for detection of S. negevensis DNA by polymerase chain reaction (PCR). RESULTS: Forty-three Crohn's disease patients and 18 controls were enrolled. Crohn's disease patients had higher prevalence of IgA antibodies to S. negevensis compared with controls (20.9% versus 0%, p = 0.04). Simkaniaceae negevensis DNA was detected in 34.9% and 5.6% of intestinal biopsies in Crohn's disease patients and controls, respectively (p = 0.02). All Crohn's disease patients with PCR-positive biopsies for S. negevensis were IgG seropositive, with specific IgA in 60% of them (p < 0.001). Immunosuppressive therapies, extraintestinal manifestations, or disease activity did not influence the presence of S. negevensis in the Crohn's disease population. CONCLUSIONS: We identified S. negevensis in Crohn's disease patients by demonstrating the presence of S. negevensis mucosal DNA and seropositivity to the bacterium. These results could support the presence of an acute or persistent S. negevensis infection and suggest a possible role in the pathogenesis of Crohn's disease.

Seroprevalence of a "new" bacterium, Simkania negevensis, in renal transplant recipients and in hemodialysis patients.

BACKGROUND: Simkania negevensis is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydiales order. It is considered an ubiquitous microorganism and aquatic environments may be involved as a source of infection for humans. It was just isolated in samples from domestic water supplies and from mains water supplies, like spa water or swimming pool water, confirming its ability to resist to the common chlorination treatments. Evidence indicates a possible role of the microorganism in respiratory tract infections, in gastroenteric disorders and in the pathogenesis of cardiovascular disease, furthermore it has hypothesized that it could play a role in lung transplant rejection. Prevalence and possible effects in nephrology are unknown. METHODS: We examined the occurrence of Simkania negevensis in two differents populations, both characterized by a high susceptibility to infectious complications: 105 hemodialysis patients, 105 renal transplant recipients and 105 healthy subjects through the IgG and IgA response to Simkania negevensis in their sera. Serum antibodies to Simkania negevensis were detected by a homemade ELISA performed according to the Kahane's protocol. Furthermore water samples from hemodialytic circuit were collected, to evaluate Simkania negevensis resistance to usual treatment of disinfection. RESULTS: Our results were unexpected, showing a higher seroprevalence of antibodies against Simkania negevensis in the hemodialysis patients, compared to renal transplant patients (IgG 22% vs 9% - IgA 9% vs 3%). S. negevensis was isolated in all water samples analyzed. CONCLUSIONS: Our study detected for the first time the occurrence of S. negevensis in hemodialysis and in renal transplant patients. Our findings suggest that water used in hemodialysis could be one of the possible sources of S. negevensis infection, without clinical involvement risk for patients.

Simkania negevensis and acute cellular rejection in lung transplant recipients.

Simkania negevensis infection has been hypothesized to play a role in lung transplant rejection. The incidence of S. negevensis infection and its association with acute cellular rejection (ACR) were determined in a prospective cohort study of 78 lung transplant recipients (LTRs) in Toronto, Canada, and Pittsburgh, USA, from July 2007 to January 2010. Simkania negevensis testing was detected by quantitative polymerase chain reaction (PCR) on bronchoalveolar lavage fluid. The relationship between S. negevensis and ACR was examined using Cox proportional hazards models and generalized linear and latent mixed models. Cumulative incidence estimates for time-to-ACR in S. negevensis PCR-positive vs. PCR-negative LTRs were 52.7% vs. 31.1% at six months and 68.9% vs. 44.6% at one yr, respectively. Although not statistically significant, there was a trend toward a higher risk of ACR among S. negevensis PCR-positive vs. PCR-negative LTRs in all statistical models.

Prevalence of Simkania negevensis in chlorinated water from spa swimming pools and domestic supplies.

AIMS: This study aimed to investigate the prevalence of Simkania negevensis in the chlorinated water of spa swimming pools and domestic network systems. METHODS AND RESULTS: A total of 10 and 36 samples were collected from two domestic water supplies and four spa facilities, respectively. Simkania negevensis was isolated in Acanthamoeba castellanii monolayers and detected by Gimenez staining and immunofluorescence test. Simkania negevensis DNA, extracted from the positive samples, was confirmed by a nested PCR assay followed by sequencing. Simkania negevensis was detected in 41·3% of samples (domestic water: 50%; untreated spa supply water: 25%; chlorinated spa pool water: 42·9%) from all the examined water systems in successive samplings performed in 1 year. The presence of S. negevensis was not correlated with the counts of Heterotrophic Bacteria and Pseudomonas aeruginosa. Unlike Legionella spp., which were never isolated from the swimming-pool water samples, S. negevensis was also detected in chlorinated spa water. CONCLUSIONS: This investigation shows that Simkania is widespread in natural and man-made aquatic environments, which may represent possible sources of infection: in the swimming pools, in particular, the aerosol generated by the water movement could increase the risk of inhalation of infected particles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evidence of the presence of S. negevensis in spa pool water. Sero-epidemiological surveys on spa users could help to clarify its transmission in this environment.

Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis.

Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.

Interaction of Chlamydiae with human macrophages.

The phylum Chlamydiae contains several members that are well-known human pathogens, like Chlamydia trachomatis and C. pneumoniae. Establishing a chronic bacterial infection requires the active evasion of the host immune response. A major arm of the innate immune defence is constituted by macrophages, which fight infections by removing bacteria and triggering an adaptive immune response. However, some pathogenic Chlamydia infect and survive in macrophages at least for a certain period of time. Therefore, macrophages can serve as vehicles for the dissemination of bacterial infections from the primary infection site via the urogenital or respiratory tract to distant sites in the body. The capacity to infect macrophages seems to depend on the chlamydial strain and the source of macrophages. In vitro infections of macrophages with C. trachomatis, C. psittaci and C. pneumoniae reveal low efficiency of infection and progeny formation, as well as failure to develop mature inclusions. In contrast, the emerging pathogen, Simkania negevensis, actively replicates in macrophages. Here we summarize the current knowledge of the intracellular and molecular key mechanisms of C. trachomatis, C. pneumoniae and S. negevensis infections in human macrophages.

Simkania negevensis: Is it a real respiratory pathogen?

Simkania negevensis, a recently discovered Chlamydia-like organism, has been associated with respiratory infections such as pneumonia, bronchiolitis and chronic obstructive pulmonary disease in children and adults. The aim of the present study was to evaluate S. negevensis in the etiology of pediatric community-acquired pneumonia, bronchiolitis and asthma exacerbation in our region. Overall, 102 patients and 46 healthy controls were included in the study. S. negevensis was investigated by real time PCR (Primer Design, UK) in nasopharyngeal swab samples. It failed to be detected in either the study or control group. In conclusion, our results suggest that S. negevensis is not an important respiratory pathogen in our region.