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1.  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
2.  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
3.  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
4.  Chlamydia pneumoniae Infection in Mice Induces Chronic Lung Inflammation, iBALT Formation, and Fibrosis 
PLoS ONE  2013;8(10):e77447.
Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5×105 CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.
PMCID: PMC3808399  PMID: 24204830
5.  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
6.  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
7.  Methicillin-Resistant Staphylococcus aureus (MRSA) Burden in Nursing Homes is Associated with Environmental Contamination of Common Areas 
Variation in MRSA prevalence across nursing homes is poorly understood. Differences in environmental cleaning may be one source of variable MRSA burden.
Prospective study of environmental contamination and cleaning quality.
10 California nursing homes.
We categorized nursing homes into two groups based upon high and low differences in MRSA point prevalence and admission prevalence (delta prevalence) from nares screenings of nursing home residents. We evaluated environmental cleaning and infection control practices by (a) culturing common area objects for MRSA, (b) assessing removal of intentionally-applied marks visible only under ultraviolet light (c) administering surveys on infection control and cleaning.
Overall, 16% (78/500) of objects were MRSA-positive, and 22% (129/577) of UV-visible marks were removed. A higher proportion of MRSA-positive objects was found in the high vs. low nursing home groups (19% vs. 10%, p=0.005). Infection control and cleaning policies varied, including the frequency of common room cleaning (mean 2.5 times daily, range 1–3) and time spent cleaning per room (mean 18 min, range 7–45). In multivariate models, MRSA-positive objects were associated with high delta prevalence nursing homes (OR=2.8, p=0.005), facilities spending less time cleaning each room (OR = 2.9, p<0.001) and facilities where common rooms were cleaned less frequently (OR =1.5, p=0.01).
We found substantial variation in MRSA environmental contamination, infection control practices, and cleaning quality. MRSA environmental contamination was associated with larger differences between MRSA point and admission prevalence, less frequent common room cleaning, and less time spent cleaning per room. This suggests that modifying cleaning practices may reduce both MRSA environmental contamination and burden among nursing homes.
PMCID: PMC3376013  PMID: 22670708
Methicillin-resistant Staphylococcus aureus (MRSA); environmental contamination; cleaning quality; infection control; long term care facility
8.  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
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.  Plasmacytoid Dendritic Cells Play a Role for Effective Innate Immune Responses during Chlamydia pneumoniae Infection in Mice 
PLoS ONE  2012;7(10):e48655.
Plasmacytoid dendritic cells (pDCs) are known for their robust antiviral response and their pro-tolerance effects towards allergic diseases and tissue engraftments. However, little is known about the role pDCs may play during a bacterial infection, including pulmonary Chlamydia pneumoniae (CP). In this study, we investigated the role of pDCs during pulmonary CP infection. Our results revealed that depletion of pDCs during acute CP infection in mice results in delayed and reduced lung inflammation, with an early delay in cellular recruitment and significant reduction in early cytokine production in the lungs. This was followed by impaired and delayed bacterial clearance from the lungs which then resulted in a severe and prolonged chronic inflammation and iBALT like structures containing large numbers of B and T cells in these animals. We also observed that increasing the pDC numbers in the lung by FLT3L treatment experimentally results in greater lung inflammation during acute CP infection. In contrast to these results, restimulation of T-cells in the draining lymph nodes of pDC-depleted mice induced greater amounts of proinflammatory cytokines than we observed in control mice. These results suggest that pDCs in the lung may provide critical proinflammatory innate immune responses in response to CP infection, but are suppressive towards adaptive immune responses in the lymph node. Thus pDCs in the lung and the draining lymph node appear to have different roles and phenotypes during acute CP infection and may play a role in host immune responses.
PMCID: PMC3485374  PMID: 23119083
11.  Nursing home characteristics associated with methicillin-resistant Staphylococcus aureus (MRSA) Burden and Transmission 
BMC Infectious Diseases  2012;12:269.
MRSA prevalence in nursing homes often exceeds that in hospitals, but reasons for this are not well understood. We sought to measure MRSA burden in a large number of nursing homes and identify facility characteristics associated with high MRSA burden.
We performed nasal swabs of residents from 26 nursing homes to measure MRSA importation and point prevalence, and estimate transmission. Using nursing home administrative data, we identified facility characteristics associated with MRSA point prevalence and estimated transmission risk in multivariate models.
We obtained 1,649 admission and 2,111 point prevalence swabs. Mean MRSA point prevalence was 24%, significantly higher than mean MRSA admission prevalence, 16%, (paired t-test, p<0.001), with a mean estimated MRSA transmission risk of 16%.
In multivariate models, higher MRSA point prevalence was associated with higher admission prevalence (p=0.005) and higher proportions of residents with indwelling devices (p=0.01). Higher estimated MRSA transmission risk was associated with higher proportions of residents with diabetes (p=0.01) and lower levels of social engagement (p=0.03).
MRSA importation was a strong predictor of MRSA prevalence, but MRSA burden and transmission were also associated with nursing homes caring for more residents with chronic illnesses or indwelling devices. Frequent social interaction among residents appeared to be protective of MRSA transmission, suggesting that residents healthy enough to engage in group activities do not incur substantial risks of MRSA from social contact. Identifying characteristics of nursing homes at risk for high MRSA burden and transmission may allow facilities to tailor infection control policies and interventions to mitigate MRSA spread.
PMCID: PMC3528666  PMID: 23095678
MRSA; Healthcare-associated infection; Long-term care; Nursing home
12.  In vitro anti-HIV-1 activity of salicylidene acylhydrazide compounds 
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.
Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.
We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.
We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
PMCID: PMC3438335  PMID: 22819150
Salicylidene acylhydrazides; HIV; microbicide; iron chelation
13.  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
14.  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
15.  Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells 
PLoS ONE  2011;6(6):e20784.
Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.
PMCID: PMC3112152  PMID: 21695198
16.  Innate Immune Responses during Respiratory Infection with a Bacterial Pathogen Induce Allergic Airway Sensitization 
The original hygiene hypothesis predicts that infections should protect against asthma, but does not account for increasing evidence that certain infections may also promote asthma development. A mechanistic reconciliation of these findings has not yet emerged. In particular, the role of innate immunity in this context is unclear.
To test whether bacterial respiratory infection causes airway sensitization towards an antigen encountered in parallel, and to elucidate the contribution of innate immune responses.
Mice were infected with different doses of Chlamydia pneumoniae (CP) followed by exposure to human serum albumin (HSA) and challenge with HSA two weeks later. Airway inflammation, immunoglobulins and lymph node cytokines were assessed. Furthermore, adoptive transfer of dendritic cells (DCs) and depletion of regulatory T-cells (Tregs) was employed.
CP-pneumonia induced sensitization towards HSA resulting in eosinophilic airway inflammation after HSA-challenge. Airway sensitization depended upon severity and timing of infection: low-dose infection and antigen exposure within 5 days of infection induced allergic sensitization, while high-dose infection or antigen exposure 10 days after infection did not. Temporal and dose-related effects reflected DC activation, and could be reproduced by adoptive transfer of HSA-pulsed lung DCs from infected mice. MyD88 deficiency in DCs abolished antigen sensitization, and depletion of Tregs prolonged the time window in which sensitization could occur.
We conclude that moderate but not severe pulmonary bacterial infection can induce allergic sensitization to inert inhaled antigens by a mechanism that requires MyD88-dependent DC activation and is controlled by Tregs.
PMCID: PMC3052793  PMID: 18774395
Asthma; allergic sensitization; adjuvant; dendritic cell; Chlamydia pneumoniae; regulatory T cells; bacterial pneumonia; allergen
17.  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
18.  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
19.  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
20.  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
21.  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
25.  The NOD/RIP2 Pathway Is Essential for Host Defenses Against Chlamydophila pneumoniae Lung Infection 
PLoS Pathogens  2009;5(4):e1000379.
Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2−/− mice compared to wild-type (WT) mice at day 3. Rip2−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1−/− and Nod2−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.
Author Summary
Chlamydophila pneumoniae (C. pneumoniae) is a common intracellular parasite that causes lung infections and contributes to several diseases characterized by chronic inflammation. Toll-like receptors expressed on the cell surface detect C. pneumoniae and mount a vigorous defense, but it is not known how the cell defends itself once the pathogen has taken up residence as a parasite. We reasoned that cytosolic pattern recognition receptors called Nods (nucleotide oligomerization domain) that detect microbes that gain entry into the cell might be involved. Using mice genetically deficient in Nod1 and Nod2 or their common downstream adaptor (Rip2), we show that in lung infection, Nod proteins are indeed essential in directing a defense against C. pneumoniae. Mice with defective Nod/Rip2-dependent signaling exhibited delayed recruitment of neutrophils, blunted production of pro-inflammatory cytokines and chemokines, and evidence of defective iNOS expression and NO production. These impaired responses led to delayed clearance of bacteria, intense persistent lung inflammation, and increased mortality. By performing bone marrow transplantation experiments and direct transfer of cells into the lungs of mice, we demonstrated that intact Nod-dependent signaling in bone marrow–derived cells was critical in the defense against C. pneumoniae. Our results indicate that Nod proteins also play an important role in host defense against C. pneumoniae. Coordinated and sequential activation of TLR and Nod signaling pathways may be necessary for an efficient immune response and host defense against C. pneumoniae.
PMCID: PMC2660273  PMID: 19360122

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