Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain

Previous studies from this laboratory provided evidence that the intracellular bacterial pathogen Chlamydophila (Chlamydia) pneumoniae is present in the late-onset Alzheimer's disease (AD) brain. Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America. Culturable organisms were detectable after one and two passages in HEp-2 cells for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy. This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae.     

Identification and characterization of putative secreted antigens from Babesia microti

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.     

Cloning and molecular characterization of cDNAs encoding three Ancylostoma ceylanicum secreted proteins

Ancylostoma ceylanicum belongs to a group of soil-transmitted helminths, which infect almost 576 mln people worldwide and are a major cause of anaemia and malnutrition. Upon contact with a permissive host, third-stage larvae (L3) residing in the environment become activated larvae (ssL3), a process associated with changes in the profile of gene expression. Ancylostoma secreted proteins (ASPs) are the major proteins secreted during larvae activation and play a crucial role in hookworm adaptation to parasitism. Here we report the cloning using RACE-PCR technique of three novel ASPs from the hookworm A. ceylanicum (Ace-asp-3, Ace-asp-4, and Ace-asp-5) and computational analysis of the protein sequences. All three proteins contain SCP (Sperm Coating Protein) domain characteristic for previously described ASP proteins. Real-time PCR analysis shows significant up-regulation of Ace-asp-3 and Ace-asp-5 expression in adult worms and correlated down-regulation in ssL3 larvae. On the other hand, expression of Ace-asp-4 was increased in ssL3 stages and decreased in adult parasites.     

Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach

Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were analyzed with several computer programs. We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M. tuberculosis with high efficiency and 90% accuracy.     

Identification and characterization of Inc766, an inclusion membrane protein in Chlamydophila abortus-infected cells

We have identified the gene product of locus 766 in the transmembrane head region (TMH/Inc-region) in the Chlamydophila abortus genome by using mass spectrometry and a monoclonal antibody that reacted with the inclusion membrane. The identified protein at 32kDa, termed Inc766, formed highly stable oligomers when solubilized in the absence of beta-mercaptoethanol. These oligomers were resistant to SDS, to heat denaturation and to 8M urea, but very sensitive to beta-mercaptoethanol, consistent with conformations resulting from protein-protein interactions stabilized through disulphide bonds. Mass spectrometry analysis of immunoprecipitated infected cell lysates indicated that a dimer at 56kDa was the most prominent form in solution. Cross-linking with DSP provided supporting evidence for the formation of oligomers in situ. Inc766 was expressed at 20-24h post infection and its localization pattern in the extra-inclusion space was common in all C. abortus strains tested. Taken together, Inc766 displays unique biochemical and cellular features not encountered in other Incs from other Chlamydiaceae species. Future studies of the particular characteristics especially the interactive properties of Inc766 should contribute to our understanding of the relationship of the different chlamydial species with their respective hosts.     

Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence.

Twenty-two mutants of Mycoplasma pneumoniae spontaneously deficient in hemadsorption were isolated. Examination of mutant protein profiles by one- and two-dimensional polyacrylamide gel electrophoresis permitted the grouping of these mutants into four classes. The largest class of mutants was deficient in four high-molecular-weight proteins (215,000, 210,000, 190,000, and 140,000). A second class of mutants lacked three proteins previously designated A, B, and C (72,000, 85,000, and 37,000, respectively). A single mutant, in addition to lacking proteins A, B, and C, was missing a fourth protein of 165,000 molecular weight. The remaining mutants exhibited protein profiles apparently identical to that of the wild-type strain. All mutant strains attached to the respiratory epithelium of hamster tracheal rings in vitro at reduced levels; however, mutants lacking proteins A, B, and C recognized only neuraminidase-insensitive receptors. None of the mutants tested produced detectable pneumonia in intranasally inoculated hamsters, although one mutant class demonstrated low-level survival in vivo.     

Limited Utility of Culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for Diagnosis of Respiratory Tract Infections

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.The etiology of respiratory tract infections can be difficult to diagnose by health care practitioners because clinical history and symptoms are usually nonspecific for most community-acquired pathogens. Mycoplasma pneumoniae and Chlamydophila pneumoniae cause up to 22% of community-acquired pneumonias and 5 to 10% of cases of tracheobronchitis, pharyngitis, laryngitis, and sinusitis (3-4, 7, 10, 12). Historically, culture has been the gold standard for diagnosis. However, cultivation of these microorganisms can prove challenging because they are fastidious and may require weeks for growth. Serology is more convenient and sensitive than culture, but results are often delayed, and false-negative test results often occur early in the course of illness. Although not standardized, nucleic acid-based tests, such as PCR, provide fast and sensitive results. While such tests are not always available on site in many medical centers, the 24- to 48-h delay in transit time may still be acceptable given the higher diagnostic yield of PCR.Despite the obvious limitations of culture, physicians continue to order this test frequently. In recent years, ARUP Laboratories has received large numbers of requests, including more than 3,000/year for M. pneumoniae and more than 1,500/year for C. pneumoniae culture. Studies focusing on the value of culture either have been small in scale or have used type strains or patient isolates rather than direct patient specimens (8, 13). An accurate and reliable diagnosis of M. pneumoniae and C. pneumoniae is important to differentiate them from other common respiratory pathogens because their treatments differ (1). Being aware of the poor sensitivity of culture for these pathogens, we found the high numbers of test requests for M. pneumoniae and C. pneumoniae culture from respiratory tract specimens to be concerning and to require further investigation. To examine more closely the utility of culture for diagnosing respiratory syndromes, we compared its performance to those of nucleic acid testing and serology for detection of M. pneumoniae and C. pneumoniae.From 2003 to 2008, microbiology results of culture, PCR, and serology performed at ARUP Laboratories for M. pneumoniae and C. pneumoniae were retrospectively reviewed with a specific focus on respiratory specimens (e.g., nasal wash, nasopharyngeal swab, bronchoalveolar lavage, tracheal aspirate, sputum, and pleural fluid) for PCR and culture. Respiratory specimens were transported either refrigerated or frozen, except for C. pneumoniae culture, for which only refrigerated specimens were transported. Additional data were collected for M. pneumoniae culture (1995 to 2003). C. pneumoniae enzyme-linked immunosorbent assay (ELISA) was performed only in 2005 to 2008, while microimmunofluorescence (MIF) was performed from 2003 to 2008. For serologic tests, data from IgM testing were collected since paired serology for IgG was rarely ordered. Subset analyses were performed for those specimens that were tested by both culture and another method. In 2008, culture-negative specimens for M. pneumoniae and C. pneumoniae were prospectively collected for PCR testing. The study protocol was approved by the University of Utah Institutional Review Board.For M. pneumoniae culture, respiratory specimens were diluted if viscous, vortexed, supplemented with amphotericin B and penicillin, and inoculated into SP-4 medium. The medium was observed daily for 21 days for a decrease in pH (a red to yellow color change). Positive cultures were confirmed by fluorescent antibody testing (Chemicon MA88285, Temecula, CA) or PCR. For C. pneumoniae, specimens were vortexed and inoculated into HEp-2 cells in McCoy medium with cycloheximide. Culture cells were passaged at 72 h into new HEp-2 vials. At day 6, cells were blind stained (Bartels Chlamydia culture confirmation reagent; Trinity Biotech, Bray, Ireland). Positive results were confirmed by a Chlamydia pneumoniae-specific reagent (Argene Inc., North Massapequa, NY) or by PCR.M. pneumoniae and C. pneumoniae PCRs were carried out using a laboratory-developed real-time assay which used manual nucleic acid extraction (Qiagen, Valencia, CA), primers and minor groove-binding hybridization probes from Epoch Biosciences (Bothell, WA), LightCycler Fast Start hybridization probe master mix (Roche, Indianapolis, IN), and the ABI HT7900 sequence detection system (Applied Biosystems, Foster City, CA). The M. pneumoniae assay targets a region of the P1 surface protein gene and has a limit of detection of <200 copies/ml. The C. pneumoniae assay targets a region of the major outer membrane protein gene and has a limit of detection of <320 copies/ml.The IgM serologic testing for M. pneumoniae was performed by ELISA (values of ≥0.96 U/liter were interpreted as positive results), and for C. pneumoniae, it was performed by either MIF or ELISA (titers of ≥1:20 and index values of ≥1.51 were interpreted as positive results, respectively).For M. pneumoniae, culture was less frequently positive than PCR or serology (P < 0.001), yielding only 10 positive results out of 24,677 specimens (Table ​(Table1).1). Of 122 paired PCR and culture results, 3 were positive by PCR and none by culture. Of 285 patients for whom both IgM serology and culture performed, 19 were positive by serology and none by culture. Of the 280 prospectively collected, culture-negative specimens, none were positive when tested by PCR.

TABLE 1.

Total numbers of samples tested and percentages positive by the various methods used for Mycoplasma pneumoniae and Chlamydophila pneumoniae diagnosis

Inhibition of lymphocyte CD3 expression by Chlamydophila pneumoniae infection

Since lymphocytes are a major immune cell besides macrophages in the development of atherosclerosis, interaction between lymphocytes and Chlamydophila pneumoniae may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae. In this regard, we examined a possible alteration of CD3 expression of human lymphocyte Molt-4 cells by C. pneumoniae infection. The expression levels of CD3 molecules of lymphocyte Molt-4 cells were significantly decreased by C. pneumoniae infection. In contrast, heat-killed C. pneumoniae as well as mock (cell lysates) did not cause any alteration of CD3 expression of the cells. Treatment of the infected cells with NS-398 (cyclo-oxyganase-2 inhibitor) or AH-23848 (EP(4) prostanoid receptor antagonist) abolished the inhibition of CD3 expression. The enhanced prostaglandin E(2) (PGE(2)) productions in the culture supernatants of infected cells were confirmed by competitive enzyme-immunosorbent assay (ELISA). C. pneumoniae infection of enriched lymphocytes from human peripheral blood mononuclear cells also induced a decrease of CD3 expression. Thus, C. pneumoniae infection of lymphocytes induces a decrease of CD3 expression mediated by possibly PGE(2) production.     

Pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae in children - comparative analysis of clinical picture

PurposeThe aim of the study was comparative analysis of clinical picture and prevalence of pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae in children.Material and MethodsThe study involved 332 children hospitalized in the 3rd Department of Paediatric, Polish Mother's Memorial Hospital – Research Institute, due to pneumonia caused by Mycoplasma pneumoniae – group I or Chlamydophila pneumonia – group II.ResultsOver 2003-2009 period there were 1870 children hospitalized due to pneumonia, of which in 332 (17.8%) the Mycoplasma pneumoniae and/or Chlamydophila pneumoniae etiology was confirmed. Mycoplasma pneumoniae, Chlamydophila pneumoniae, and mixed infection was diagnosed in 198 (10.6%), 102 (5.5%), and 32 (1.7%) children, respectively. The dominant clinical feature in both groups was cough, observed in 186 (93.9%) and 88 (86.3%) children, respectively. Further, reddening of the throat, rhinitis, shortness of breath, fever, enlarged lymph nodes, skin lesions and dyspepsia were also observed. The frequency of specific clinical features in both groups was similar. Statistical relationship (p≤0.05) was observed only in case of skin lesions. In chest x-ray there was no statistical link as for analyzed changes. Interstitial inflammatory changes were most frequently observed.ConclusionsMycoplasma pneumoniae and Chlamydophila pneumoniae are significant etiological factors in pneumonia in children, and as such they should be taken into consideration in differential diagnosis of pneumonia in children. The clinical picture of pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae is hardly specific, with basic labs and chest x-ray of little help in differentiation of infection etiology.     

Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1

Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.     

Identification and characterization of immunogenic proteins of mycoplasma genitalium

Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.     

Identification and characterization of Campylobacter jejuni outer membrane proteins

Outer membrane proteins from isolates of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations were outer membrane enriched based on increased ketodeoxyoctonate concentrations, the presence of surface-exposed 125I-labeled proteins that were hydrophobic, and similarity to membrane vesicle (bleb) sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Most isolates contained a single major band with molecular weight of 41,000 to 45,000. Profiles of C. jejuni and Campylobacter coli isolates were indistinguishable, but either could be easily differentiated from Campylobacter fetus and Campylobacter faecalis. The profiles were stable for strains under a variety of growth, incubation and passage conditions. We classified 110 isolates from patients with sporadic campylobacter enteritis into nine subtypes based on differences in outer membrane sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Two categories accounted for 76% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases. Thus, comparison of the major outer membrane proteins of C. jejuni is a useful technique for investigating the transmission of this organism and may provide a basis for immunological characterization of the outer membrane proteins.     

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.     

Identification and characterization of human surfactant protein A binding protein of Mycoplasma pneumoniae

Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca(2+))-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca(2+) enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.     

Identification and characterization of structural proteins of orchid fleck virus

Orchid fleck virus (OFV) has a bipartite negative-sense RNA genome with sequence similarities to plant rhabdoviruses. The non-enveloped bullet-shaped particles of OFV are similar to those of the internal ribonucleoprotein (RNP)-M protein structure of rhabdoviruses, but they are about half the size of typical plant rhabdoviruses. Purified preparations contained intact bullet-shaped and filamentous particles. The filamentous particles showed a tightly coiled coil structure or a coiled structure with a helical twist, which resembles the RNP complex of rhabdoviruses. OFV bullet-shaped particles were structurally stable in solutions containing 2% Triton X-100 and 0.8 M NaCl. Western blot analyses revealed that the bullet-shaped particles contained N, P and M proteins, while filamentous particles contained mainly N and P proteins. In addition, a small amount of the L protein was detected in both types of particles. Thus, the structural proteins of OFV have properties similar to those of rhabdoviruses, except that the particles are non-enveloped and are relatively resistant to detergent-treatment under high-salt conditions.     

Choice of antimicrobial drug for infections caused by Chlamydia trachomatis and Chlamydophila pneumoniae

Chlamydia (C.) trachomatis is the most common bacterial cause of sexually transmitted disease in the world. A well documented feature of chlamydial infection is its high rate of recurrence among sexually active populations. However, it is difficult to distinguish whether the high rate of recurrent disease is due to reinfection or to persistent infection with the same organism. Of particular concern in this era of increasing antibiotic resistance is whether persistent infection is the consequence of increasing resistance to standard antimicrobial therapy. Azithromycin and doxycycline are considered by the Centers for Disease Control and Prevention (CDC) as first line drugs for the treatment of chlamydial infections; erythromycin, ofloxacin and levofloxacin are recommended as alternative-regimen. Although C. trachomatis has been historically sensitive to these antibiotics, in vitro resistance is being increasingly reported. However, although in vitro antimicrobial resistance has been described, the clinical significance of these findings is unknown. C. pneumoniae is associated with community-acquired pneumonias, acute exacerbations of chronic bronchitis, otitis media, sinusitis and reactive airway disease. Persistent nasopharyngeal infection with C. pneumoniae has been documented in adults following acute respiratory infection. Chronic infection with C. pneumoniae has also been implicated in the pathogenesis of atherosclerosis, although this is still very controversial. Azithromycin, clarithromycin and quinolones are frequently used for the treatment of C. pneumoniae respiratory infections. Microbiologic failure has been described in C. pneumoniae infections, even after prolonged courses of azithromycin, erythromycin and doxycycline.     

Identification of a novel Citrobacter rodentium type III secreted protein, EspI, and roles of this and other secreted proteins in infection

Citrobacter rodentium is a member of a group of pathogens that colonize the lumen of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. C. rodentium, which causes transmissible colonic hyperplasia in mice, is used as an in vivo model system for the clinically significant A/E pathogens enterohemorrhagic and enteropathogenic Escherichia coli. These bacteria all contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III secretion system that is designed to deliver effector proteins into eukaryotic host cells. These effectors are involved in the subversion of host eukaryotic cell functions to the benefit of the bacterium. In this study we used mutant strains to determine the effects of the C. rodentium LEE-encoded effectors EspF, EspG, EspH, and Map on virulence in the mouse model. In addition, we identified a novel secreted protein, EspI encoded outside the LEE, whose secretion is also dependent on a functional type III secretion system. Mutant strains with each of the effectors investigated were found to be outcompeted by wild-type bacteria in mixed-infection experiments in vivo, although the effects of EspF and EspH were only subtle. In single-infection experiments, we found that EspF, EspG, and EspH are not required for efficient colonization of the mouse colon or for the production of hyperplasia. In contrast, strains producing EspI and Map had significant colonization defects and resulted in dramatically reduced levels of hyperplasia, and they exhibited very different growth dynamics in mice than the wild-type strain exhibited.     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.