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1.  Comparison of Broth Enhancement to Direct Plating for Screening of Rectal Cultures for Ciprofloxacin-Resistant Escherichia coli 
Journal of Clinical Microbiology  2013;51(1):249-252.
A transrectal prostate biopsy is the most common procedure used to establish the diagnosis of prostate cancer. Prior to biopsy, patients are commonly given ciprofloxacin for prophylaxis. However, a complication of the procedure is infection with ciprofloxacin-resistant organisms, in particular resistant Escherichia coli. In order to identify patients carrying ciprofloxacin-resistant E. coli, so as to tailor their antibiotic prophylaxis, rectal swabs are screened using selective broth and/or solid medium. In our evaluation, we compared broth enrichment and direct plating techniques by using brain heart infusion broth and MacConkey agar containing 1 μg/ml or 10 μg/ml of ciprofloxacin. Of the 100 patients included in the study, 20 were colonized with ciprofloxacin-resistant organisms, 19 of which were E. coli. There was no significant difference (P > 0.1) between the culture methods or the ciprofloxacin concentrations in the medium when identifying patients with ciprofloxacin-resistant E. coli; however, broth enrichment using 1 μg/ml ciprofloxacin was the most sensitive at 100%, but it was the least specific. Direct plating of rectal swabs onto MacConkey agar containing 10 μg/ml of ciprofloxacin was 100% specific and missed only 1 positive specimen, with a sensitivity of 94.7%; this method was the most cost-effective. Therefore, direct plating of rectal swabs onto selective medium proved to be a sensitive and cost-effective approach in identifying patients colonized with ciprofloxacin-resistant E. coli.
PMCID: PMC3536248  PMID: 23152550
2.  Comparison of Medium, Temperature, and Length of Incubation for Detection of Vancomycin-Resistant Enterococcus 
Journal of Clinical Microbiology  2012;50(7):2503-2505.
Campylobacter (Campy; BD Diagnostics, Sparks, MD), Spectra VRE (Remel, Lenexa, KS), and bile-esculin-azide-vancomycin (BEAV; Remel) agars were compared for their ability to detect vancomycin-resistant enterococci (VRE) in 750 stool specimens. The media were compared at 24 h and 48 h of incubation at 35°C and 42°C. When incubated for 24 h at 35°C, Campy was the most sensitive (97.8%) and specific (99.9%) but was comparable to Spectra, which has a sensitivity of 95.6% and a specificity of 99.1%, whereas BEAV was significantly less sensitive (90%) and specific (96.1%). Incubation at 42°C or extended incubation at 35°C for 48 h yielded no advantage over incubation at 35°C for 24 h.
PMCID: PMC3405587  PMID: 22535989
3.  Formulation of the Microbicide INP0341 for In Vivo Protection against a Vaginal Challenge by Chlamydia trachomatis 
PLoS ONE  2014;9(10):e110918.
The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.
PMCID: PMC4214720  PMID: 25356686
4.  Dendritic Cells from Aged Subjects Display Enhanced Inflammatory Responses to Chlamydophila pneumoniae 
Mediators of Inflammation  2014;2014:436438.
Chlamydophila pneumoniae (CPn) is a common respiratory pathogen that causes a chronic and persistent airway infection. The elderly display an increased susceptibility and severity to this infection. However, the underlying mechanisms are not well understood. Dendritic cells (DCs) are the initiators and regulators of immune responses. Therefore, we investigated the role of DCs in the age-associated increased CPn infection in vitro in humans. Though the expression of activation markers was comparable between the two age groups, DCs from aged subjects secreted enhanced levels of proinflammatory mediators such as TNF-α and CXCL-10 in response to CPn. In contrast, the secretion of IL-10 and innate interferons, IFN-α and IFN-λ, was severely impaired in DCs from aged donors. The increased activation of DCs from aged subjects to CPn also resulted in enhanced proliferation of CD4 and CD8 T cells in a DC-T coculture. Furthermore, T cells primed with CPn-stimulated DCs from aged subjects secreted increased levels of IFN-γ and reduced levels of IL-10 compared to DCs obtained from young subjects. In summary, DCs from the elderly displayed enhanced inflammatory response to CPn which may result in airway remodeling and increase the susceptibility of the elderly to respiratory diseases such as asthma.
PMCID: PMC4165882  PMID: 25253920
5.  Prevalence of ST131 Among Fluoroquinolone-resistant Escherichia coli Obtained From Rectal Swabs Before Transrectal Prostate Biopsy 
Urology  2013;81(3):548-555.
To identify the prevalence and characteristics of fluoroquinolone-resistant (FQ-R) Escherichia coli ST131 isolates in men undergoing ultrasound-guided transrectal prostate biopsy (TPB).
Twenty-seven FQ-R E coli isolates from rectal swabs from 136 men undergoing TPB at 3 institutions in southern California (January 2009 to March 2010), with a focus on repeat biopsy patients, were assessed for E coli phylogenetic group, sequence type ST131 status, extended virulence genotype, pulsed-field gel electrophoresis profile, and antimicrobial susceptibility profile.
ST131 accounted for 70% of the 27 FQ-R pre-TPB E coli rectal isolates, including 82% of those from non-Asians vs 20% from Asians (P = .017). ST131 was associated negatively with prebiopsy enemas and positively with previous TPB. Compared with non-ST131 isolates, the ST131 isolates had a significantly higher prevalence of 4 virulence genes (sat, usp, ompT, and malX), distinctive virulence profiles, and numerically higher virulence scores (median, 12 vs 8), but similar antimicrobial resistance scores. Most rectal ST131 isolates exhibited pulsed-field gel electrophoresis profiles typical of clinical ST131 isolates.
In our locale, the epidemic multidrug-resistant ST131 clonal group accounts for 70% of FQ-R rectal E coli isolates among men undergoing TPB. Such ST131 isolates have distinctive virulence profiles, are extensively antimicrobial-resistant, and are negatively associated with Asian race. Further investigation is needed regarding risk factors for and clinical consequences of colonization with such strains among men undergoing TPB.
PMCID: PMC4066977  PMID: 23333000
6.  Prevalence and Significance of Fluoroquinolone Resistant Escherichia coli in Patients Undergoing Transrectal Ultrasound Guided Prostate Needle Biopsy 
The Journal of urology  2011;185(4):1283-1288.
We estimated the prevalence of fluoroquinolone resistant Escherichia coli in patients undergoing repeat transrectal ultrasound guided prostate needle biopsy and identified high risk groups.
Materials and Methods
From January 2009 to March 2010 rectal swabs of 136 men from 3 institutions undergoing transrectal ultrasound guided prostate needle biopsy were obtained. There were 33 men with no previous biopsy who served as the controls. Participants completed questionnaires and rectal swab culture was obtained just before performing the prostate biopsy. Selective media was used to specifically isolate fluoroquinolone resistant E. coli and sensitivities were obtained. The patients were contacted via telephone 7 days after the procedure for a followup questionnaire.
A total of 30 patients had cultures positive for fluoroquinolone resistant bacteria for an overall rate of 22% (95% CI 15, 29). Patients with diabetes and Asian ethnicity had higher risks of resistant rectal flora colonization (OR 2.3 and 2.8, respectively). However, differences did not reach statistical significance (p = 0.09 and p = 0.08, respectively). Patients with no prior biopsy had a positive rate of 15% (5 of 33) compared to 24% (25 of 103) in those with 1 or more prior biopsies (OR 1.8, p = 0.27). Five patients (3.6%) had post-biopsy fever while only 1 of those patients had a positive rectal swab.
Using selective media to isolate fluoroquinolone resistant E. coli from the rectum before transrectal ultrasound guided prostate biopsy, we isolated organisms in 22% of patients with a wide resistance pattern. This protocol may be used to provide information regarding targeted antibiotic prophylaxis before transrectal prostate biopsies.
PMCID: PMC4063558  PMID: 21334021
prostate; biopsy; drug resistance; microbial; fluoroquinolones; postoperative complications
7.  Screening Rectal Culture to Identify Fluoroquinolone-resistant Organisms Before Transrectal Prostate Biopsy: Do the Culture Results Between Office Visit and Biopsy Correlate? 
Urology  2013;82(1):67-71.
To investigate the performance of screening rectal cultures obtained 2 weeks before transrectal prostate biopsy to detect fluoroquinolone-resistant organisms and again at transrectal prostate biopsy.
After institutional review board approval for observational study, we obtained a rectal culture on patients identified for a prostate biopsy but before antibiotic prophylaxis from September 12, 2011 to April 23, 2012. The specimen was cultured onto MacConkey agar with and without 1 µg/mL ciprofloxacin. We then obtained a second rectal culture immediately before prostate biopsy after 24 hours of ciprofloxacin prophylaxis. All cultures were blinded to the practitioner until the end of the study.
Of 108 patients enrolled, 58 patients had both rectal cultures for comparison. The median time duration between cultures was 14 (6–119) days. There were 54 of 58 concordant pairs (93%), which included 47 negative cultures and 7 positive cultures; 2 patients (3%) who were culture negative from the first screening culture became positive at biopsy. Sensitivity, specificity, negative, positive predictive values, and area under the operator curve were 95.9%, 77.8%, 95.9%, 77.8%, and 0.868, respectively. When Pseudomonas spp. are removed from the analysis, the area under the curve is increased to 0.927.
Screening rectal cultures 2 weeks before prostate biopsy has favorable test performance, suggesting screening cultures give an accurate estimate of fluoroquinolone-resistant colonization.
PMCID: PMC3952011  PMID: 23806391
8.  Chlorhexidine and Mupirocin Susceptibilities of Methicillin-Resistant Staphylococcus aureus from Colonized Nursing Home Residents 
Chlorhexidine and mupirocin are used in health care facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The objective of this study was to assess the frequency of chlorhexidine and mupirocin resistance in isolates from nares carriers in multiple nursing homes and to examine characteristics associated with resistance. Nasal swab samples were collected from approximately 100 new admissions and 100 current residents in 26 nursing homes in Orange County, CA, from October 2008 to May 2011. MRSA isolates were tested for susceptibility by using broth microdilution, disk diffusion, and Etest; for genetic relatedness using pulsed-field gel electrophoresis; and for qac gene carriage by PCR. Characteristics of the nursing homes and their residents were collected from the Medicare Minimum Data Set and Long-Term Care Focus. A total of 829 MRSA isolates were obtained from swabbing 3,806 residents in 26 nursing homes. All isolates had a chlorhexidine MIC of ≤4 μg/ml. Five (0.6%) isolates harbored the qacA and/or qacB gene loci. Mupirocin resistance was identified in 101 (12%) isolates, with 78 (9%) isolates exhibiting high-level mupirocin resistance (HLMR). HLMR rates per facility ranged from 0 to 31%. None of the isolates with HLMR displayed qacA or qacB, while two isolates carried qacA and exhibited low-level mupirocin resistance. Detection of HLMR was associated with having a multidrug-resistant MRSA isolate (odds ratio [OR], 2.69; P = 0.004), a history of MRSA (OR, 2.34; P < 0.001), and dependency in activities of daily living (OR, 1.25; P = 0.004). In some facilities, HLMR was found in nearly one-third of MRSA isolates. These findings may have implications for the increasingly widespread practice of MRSA decolonization using intranasal mupirocin.
PMCID: PMC3535956  PMID: 23147721
9.  Protection of Mice From a Chlamydia trachomatis Vaginal Infection Using a Salicylidene Acylhydrazide, a Potential Microbicide 
The Journal of Infectious Diseases  2011;204(9):1313-1320.
The salicylidene acylhydrazide INP0341 inhibits growth of Chlamydia in HeLa cells, has negligible cell toxicity, and does not inhibit the growth of lactobacilli. The antichlamydial activity of INP0341 was retained when tested in vaginal and semen simulants. Vaginal tissue from INP0341-treated mice appeared similar to control sham-treated mice. To determine whether INP0341 can protect mice from a vaginal challenge, C3H/HeJ mice were either sham or INP0341 treated intravaginally pre- and postinoculation with 5 × 102 inclusion-forming units (IFUs) of Chlamydia trachomatis serovar D. Vaginal cultures taken over a month-long period showed a significant difference in the number of control mice that were culture positive versus the number in the INP0341-treated group, 100% (25/25) and 31% (8/26), respectively (P < .05). The quantity of IFUs shed and antibody titers to Chlamydia were significantly higher for the control group (P < .05). In summary, INP0341 is a promising compound to be considered for formulation as a vaginal microbicide.
PMCID: PMC3182314  PMID: 21933873
10.  Detection of Fluoroquinolone-Resistant Organisms from Rectal Swabs by Use of Selective Media Prior to a Transrectal Prostate Biopsy▿ 
Journal of Clinical Microbiology  2011;49(3):1116-1118.
Sepsis caused by fluoroquinolone-resistant Escherichia coli is a risk for patients undergoing an ultrasound-guided, transrectal prostate biopsy. A method incorporating selective broth and media was evaluated using rectal swabs obtained from 136 patients prior to a biopsy procedure. Fluoroquinolone-resistant organisms were isolated from 22% of the patients included in this study.
PMCID: PMC3067719  PMID: 21177893
11.  Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseria gonorrhoeae 
Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis serovar L2, Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 µM towards C. trachomatis L2. Three INPs had an MIC <12.5 µM against N. gonorrhoeae. Inhibition by was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection (P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.
PMCID: PMC2902681  PMID: 20605703
Vaginal microbicide; Sexually transmitted infections; Chlamydia trachomatis; LGV; Neisseria gonorrhoeae
12.  Promoters for Chlamydia Type III Secretion Genes Show a Differential Response to DNA Supercoiling That Correlates with Temporal Expression Pattern▿  
Journal of Bacteriology  2010;192(10):2569-2574.
Type III secretion (T3S) is important for the establishment and maintenance of a chlamydial infection. The genes encoding T3S components in Chlamydia are transcribed as separate temporal classes, but the mechanisms that regulate the timing of their expression are not understood. In this study, we demonstrate that promoters for 10 predicted T3S transcriptional units are each transcribed in vitro by the major form of chlamydial RNA polymerase but not by an alternative form of RNA polymerase containing σ28. Since changes in DNA supercoiling during chlamydial development have been proposed as a mechanism for temporal gene regulation, we examined the in vitro response of T3S promoters to altered superhelical density. Promoters for three T3S genes that are upregulated at mid times were activated in response to increased DNA supercoiling. In contrast, promoters for three late T3S genes were not sensitive to changes in superhelical density. This differential response to changes in DNA topology is similar to the pattern that has been reported for representative mid and late chlamydial genes that are unrelated to the T3S system. Based on these results, we propose that the temporal expression of T3S genes in Chlamydia is controlled by general mechanisms that regulate σ66-dependent gene expression during the developmental cycle. Our results are consistent with a model in which T3S genes that are upregulated in mid cycle are activated together with other mid genes in response to increased DNA supercoiling.
PMCID: PMC2863571  PMID: 20233926
13.  Protection against an intranasal challenge by vaccines formulated with native and recombinant preparations of the Chlamydia trachomatis major outer membrane protein 
Vaccine  2009;27(36):5020-5025.
To compare the ability of a native and a recombinant preparation of the major outer membrane protein of Chlamydia trachomatis mouse pneumonitis (MoPn; Ct-nMOMP and Ct-rMOMP) to protect against an intranasal (i.n.) challenge, BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-1826 and Montanide ISA 720 as adjuvants. Animals inoculated i.n. with live elementary bodies (EB) of Chlamydia served as a positive control. Negative control groups were immunized with either Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or with minimal essential medium (MEM-0). Mice immunized with Ct-rMOMP, Ct-nMOMP and EB developed a strong immune response as shown by high levels of Chlamydia specific antibodies in serum and a strong T-cell lymphoproliferative response. Following the i.n. challenge with 104 inclusion forming units (IFU) of C. trachomatis. mice immunized with Ct-nMOMP or Ct-rMOMP lost significantly less weight than the negative control animals immunized with Ng-rPorB or MEM-0 (P<0.05). However, mice vaccinated with the Ct-nMOMP lost less weight than those immunized with the Ct-rMOMP (P<0.05). Mice were euthanized at 10 days following the challenge, their lungs weighed and the number of IFU of Chlamydia determined. Based on the lung weight and number of IFU recovered, significant protection was observed in the groups of mice immunized with both Ct-nMOMP and the Ct-rMOMP (P<0.05). Nevertheless, significantly better protection was achieved with the Ct-nMOMP in comparison with the Ct-rMOMP (P<0.05). In conclusion, vaccination with a preparation of the nMOMP elicited a more robust protection than immunization with rMOMP suggesting that the conformational structure of MOMP is critical for inducing strong protection.
PMCID: PMC2741729  PMID: 19446590
Chlamydia trachomatis; vaccines; major outer membrane protein; mice
14.  Chlamydia trachomatis Native Major Outer Membrane Protein Induces Partial Protection in Non-Human Primates: Implication for a Trachoma Transmission Blocking Vaccine 
A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a non-human primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA antibody titers, with antibodies displaying high strain-specific neutralizing activity. The PBMC of immunized monkeys produced a broadly cross-reactive, antigen-specific IFN-γ response equivalent to that induced by experimental infection. Immunized monkeys exhibited a highly significant decrease in infectious burden during the early peak shedding periods (days 3-14). However, at later time points they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in non-human primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a marked, significant reduction in ocular shedding in non-human primates. A partially protective vaccine, particularly one that significantly reduces infectious burden following primary infection of children, could interrupt the natural trachoma re-infection cycle. This could have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.
PMCID: PMC2692073  PMID: 19494332
Vaccination; Mucosa; Bacterial; Antigens/Peptides/Epitopes; Other Animals
15.  TLR/MyD88 and LXRα Signaling Pathways Reciprocally Control Chlamydia Pneumoniae-Induced Acceleration of Atherosclerosis 
Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and athero-thrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in ApoE−/− mice is reciprocally modulated by activation of TLR-mediated innate immune or LXRα signaling pathways. We infected ApoE−/− mice and ApoE−/− mice that also lacked TLR2 or TLR4 or MyD88 or LXRα intranasally with C. pneumoniae followed by high-fat diet feeding for 4 months. Mock infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques, and serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6 and TNF-α. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE−/− mice that were also deficient in either TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE−/− mice was further enhanced in ApoE−/−/LXRα−/− double knockout mice, and was accompanied by higher serum levels of IL-6 and TNF-α. We conclude that C. pneumoniae-infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism, and that LXRα appears to reciprocally modulate and reduce the pro-atherogenic effects of C. pneumoniae infection.
PMCID: PMC2683843  PMID: 18981139
16.  Protection of Wild-Type and Severe Combined Immunodeficiency Mice against an Intranasal Challenge by Passive Immunization with Monoclonal Antibodies to the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein▿  
Infection and Immunity  2008;76(12):5581-5587.
Monoclonal antibodies (MAbs) to the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) were characterized for their ability to neutralize the infectivity of this organism in vitro and in vivo. One of the MAbs (MoPn-23) recognizes a nonlinear epitope in the MOMP, MAb MoPn-40 binds to a linear epitope in the variable domain 1 (VD1), and MAb MoPn-32 recognizes the chlamydial lipopolysaccharide. MAb MoPn-23 neutralized 50% of the infectivity of Chlamydia, as measured in vitro by using HAK (FcγIII−) and HeLa-229 (FcγIII+) cells at a concentration 100 times lower than MAb MoPn-40. MAb MoPn-32 had no neutralizing ability. In comparison to the control normal mouse immunoglobulin G, passive immunization of BALB/c mice with MAb MoPn-23 resulted in a highly significant protection against an intranasal (i.n.) challenge as determined by the change in body weight, the weight of the lungs, and the yield of Chlamydia inclusion-forming units (IFU) from the lungs. Passive immunization with MAb MoPn-40 resulted in a lower degree of protection, and MAb MoPn-32 afforded no protection. MAb MoPn-23 was also tested for its ability to protect wild-type (WT) and severe combined immunodeficient (SCID) C.B-17 mice against an i.n. challenge. Protection based on total body weight, lung weight, and yield of Chlamydia IFU was as effective in SCID as in WT C.B-17 mice. In conclusion, antibodies to MOMP can protect mice against a chlamydial infection in the presence or absence of T and B cells.
PMCID: PMC2583570  PMID: 18809664
17.  Structural and Functional Analyses of the Major Outer Membrane Protein of Chlamydia trachomatis▿  
Journal of Bacteriology  2007;189(17):6222-6235.
Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of β-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37°C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a β-sheet structure and porin function.
PMCID: PMC1951919  PMID: 17601785
18.  Reversal of the Antichlamydial Activity of Putative Type III Secretion Inhibitors by Iron▿  
Infection and Immunity  2007;75(7):3478-3489.
INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 μM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of ≥20 μM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.
PMCID: PMC1932962  PMID: 17470544
19.  Vaccination with the Chlamydia trachomatis Major Outer Membrane Protein Can Elicit an Immune Response as Protective as That Resulting from Inoculation with Live Bacteria  
Infection and Immunity  2005;73(12):8153-8160.
BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes with a native preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP), using Montanide ISA 720 and CpG-1826 as adjuvants. A negative control group was immunized with ovalbumin and the two adjuvants, and a positive control group was immunized intranasally (i.n.) with 104 inclusion-forming units (IFU) of C. trachomatis. Four weeks after the last i.m.-plus-s.c. immunization, mice were challenged in the ovarian bursa with 105 IFU of C. trachomatis MoPn. Six weeks after the genital challenge, animals were mated, and the pregnancies were monitored. After vaccination with MOMP, the mice developed strong Chlamydia-specific humoral and cellular immune responses. Following the genital challenge, of the mice vaccinated with the MOMP, only 15% (3/20) had positive vaginal cultures, while 85% (17/20) of the animals immunized with ovalbumin had positive cultures over the 6 weeks of observation (P < 0.05). Also, only 14% (3/21) of the animals inoculated i.n. with Chlamydia had positive vaginal cultures. After mating, 75% (15/20) of the mice vaccinated with MOMP carried embryos in both uterine horns. Of the animals vaccinated i.n. with the Chlamydia, 81% (17/21) had embryos in both uterine horns (P > 0.05). In contrast, only 10% (2/20) of the mice immunized with ovalbumin had embryos in both uterine horns (P < 0.05). In conclusion, immunization with a purified preparation of the MOMP is as effective as vaccination with viable C. trachomatis in eliciting a protective immune response against a genital challenge in mice.
PMCID: PMC1307068  PMID: 16299310
20.  CHROMagar Candida Medium for Direct Susceptibility Testing of Yeast from Blood Cultures 
Journal of Clinical Microbiology  2005;43(4):1727-1731.
An evaluation was performed on 95 blood cultures positive for Candida spp. to determine the correlation of direct susceptibility testing of fluconazole versus both standardized disk diffusion and MIC methods. For direct testing, an aliquot taken from BD BACTEC Plus and/or BD BACTEC Lytic/10 bottles (Becton Dickinson [BD], Sparks, MD) positive by gram stain for yeast was subcultured to CHROMagar Candida (BD), and a 25-μg fluconazole disk (BD) was placed on the plate. The area of growth inhibition surrounding the disk was measured at 24 and 48 h. In addition, a subculture of the isolate was tested by a microdilution MIC using YeastOne (TREK Diagnostics Systems Inc., OH) and disk diffusion (NCCLS M44-A) using a standardized inoculum plated onto CHROMagar Candida as well as Mueller-Hinton agar to which 2% glucose and 0.5 μg/ml methylene blue dye was added (MH-GMB). The categorical interpretation derived from the MIC was used as the reference to which the disk diffusion results were compared. There were a total of 41 Candida albicans, 23 Candida glabrata, 20 Candida parapsilosis, 9 Candida tropicalis, and 1 each of Candida krusei and Candida lusitaniae tested. At 24 h there was full agreement among the methods for all C. albicans, C. tropicalis, C. lusitaniae, and C. krusei isolates. For the C. parapsilosis isolates at 24 h there was one very major discrepancy using the direct CHROMagar and one major error with the standardized MH-GMB. The majority of the errors were seen at 24 h with the C. glabrata isolates. Of the 23 C. glabrata isolates at 24 h by direct CHROMagar, there were 10 minor and 1 very major error; by MH-GMB there were 12 minor and 2 very major errors; and by standardized CHROMagar Candida there were 13 minor and 2 major errors. There were no very major errors with C. glabrata when all plates were read at 48 h. At 24 h by the direct and standardized CHROMagar the majority of C. glabrata isolates were more resistant, whereas by MH-GMB they were more susceptible than the reference MIC interpretation. In summary, subculturing yeast directly from blood cultures onto CHROMagar to which a fluconazole disk has been added may provide a presumptive identification at 24 h and, with the exception of C. glabrata, was able to predict the susceptibility to fluconazole with the majority of Candida isolates examined in this evaluation.
PMCID: PMC1081328  PMID: 15814992
21.  Interaction between Components of the Type III Secretion System of Chlamydiaceae 
Journal of Bacteriology  2005;187(2):473-479.
Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as “bait” to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.
PMCID: PMC543553  PMID: 15629918
22.  New Murine Model for the Study of Chlamydia trachomatis Genitourinary Tract Infections in Males  
Infection and Immunity  2004;72(7):4210-4216.
The lack of an experimental model has significantly limited the understanding of the pathogenesis of Chlamydia trachomatis infections in males. In an attempt to establish a model using the natural route of infection, we inoculated male mice in the meatus urethra. To establish the 50% infectious dose (ID50), C3H/HeN (H-2k) male mice were inoculated in the meatus urethra with doses ranging from 101 to 107 inclusion-forming units (IFU) of C. trachomatis mouse pneumonitis biovar (MoPn) and were euthanized at 10 days postinfection (p.i.). Approximately 50% of the animals inoculated with 5 × 104 IFU had positive cultures of the urethra, urinary bladder, epididymides, and/or testes. Subsequently, to characterize the course of the infection, a group of animals was inoculated with 106 IFU/mouse (20 times the ID50). Positive cultures from the urethra, urinary bladder, epididymides, and testes were obtained from the animals. The infection peaked in the first 2 weeks p.i. and subsequently declined over the 7 weeks of observation. C. trachomatis-specific antibodies were first detected in serum by 2 weeks p.i. and rose over the period of observation. The titers of immunoglobulin G2a (IgG2a) were 16-fold higher than those of IgG1. A lymphoproliferative assay using splenocytes and local lymph nodes showed a strong cell-mediated immune response. Levels of gamma interferon were significantly higher than those of interleukin-4 in the supernatants from stimulated lymphocytes. An acute inflammatory infiltrate consisting of polymorphonuclear leukocytes was detected in the urethra at 1 week p.i. At 3 weeks p.i., a mixed acute and chronic inflammatory infiltrate was observed in the urethra that by 5 to 6 weeks was mainly composed of mononuclear cells. Similar findings were also observed in the urinary bladder, although the inflammatory infiltrate was delayed by approximately a week relative to that in the urethra. Sections of the epididymides showed a focal acute inflammatory infiltrate at 2 weeks p.i. Immunohistochemical staining demonstrated multiple chlamydial inclusions in the epithelium of the urethra and urinary bladder. No chlamydial inclusions were observed in the epididymides or testes. In conclusion, inoculation of male mice in the meatus urethra with C. trachomatis MoPn results in an infection of the genitourinary tract that closely parallels that described in humans. This model should help to characterize the pathogenesis of chlamydial infections in males and to test therapeutic and preventive measures.
PMCID: PMC427456  PMID: 15213165
23.  Temporal Expression of Type III Secretion Genes of Chlamydia pneumoniae  
Infection and Immunity  2003;71(5):2555-2562.
Chlamydia pneumoniae has been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems. Upon infection of HEp-2 cells with C. pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen. In addition, expression was analyzed when C. pneumoniae was grown in the presence of human gamma interferon (IFN-γ). The groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of the fstK gene was used as a marker for the effect of IFN-γ on the maturation of C. pneumoniae. In the absence of IFN-γ, the TTS genes were expressed as follows: early stage (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2; middle stage (by 12 to 18 h), lcrD, yscN, and yscR; and late stage (by 24 h), lcrE, sycE, lcrH-1, and yscT. Of the genes expressed early, the lcrH-2 gene was detected the earliest, at 1.5 h. Expression of the yscU gene was not detected at any of the time points examined. Under the influence of IFN-γ, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely, lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated, and expression could not be detected up to 48 h. In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-γ. The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to IFN-γ. In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell. In summary, the expression pattern of the TTS genes of C. pneumoniae examined suggests that they are temporally regulated throughout the developmental cycle. Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-γ.
PMCID: PMC153279  PMID: 12704128
24.  Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein by Use of CpG Oligodeoxynucleotides as an Adjuvant Induces a Protective Immune Response against an Intranasal Chlamydial Challenge  
Infection and Immunity  2002;70(9):4812-4817.
Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 104 inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.
PMCID: PMC128273  PMID: 12183524
25.  Inclusion Fluorescent-Antibody Test as a Screening Assay for Detection of Antibodies to Chlamydia pneumoniae 
A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the “gold standard.” In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of ≥16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of ≥512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (≥16) or elevated (≥512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).
PMCID: PMC119991  PMID: 11986260

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